Method for the analysis of glycosaminoglycans, and their derivatives and salts by nuclear magnetic resonance

ABSTRACT

An analytical method employing nuclear magnetic resonance of glycosaminoglycans in general, and of heparins and low molecular weight heparins and their derivatives in particular, is provided. The method is used for identification and the relative quantification of characteristic signals by 1H-NMR and/or 1H-13C HSQC.

CROSS-REFERENCE TO EARLIER FILED APPLICATIONS

The present application claims the benefit of and is acontinuation-in-part of international application PCT/EP2017/068285filed Jul. 19, 2017, which claims the benefit of European application EP16382350.3 filed Jul. 19, 2016, the entire disclosures of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention concerns an analytical method employing nuclearmagnetic resonance (¹H-NMR, ¹³C-NMR, and/or ¹H-¹³C-HSQC) for thecharacterization of glycosaminoglycans (GAG's), derivatives thereof,and/or salts thereof. The method allows for qualitative and quantitativeanalysis of the saccharides comprising said GAG's.

BACKGROUND OF THE INVENTION

Nuclear magnetic resonance (NMR) spectroscopy is one of the mostimportant and widespread analytical techniques used in thecharacterization of glycosaminoglycans in general, and heparins and lowmolecular weight heparins and their derivatives in particular.

The possibility of performing both one-dimensional and two-dimensionalexperiments makes this technique highly sensitive for determining smallvariations in molecular structure, making it very advantageous for asuitable characterization of these compounds.

Glycosaminoglycans (GAGs) are linear and negatively chargedpolysaccharides with a mean molecular weight between 10-100 KDa (“TheStructure of Glycosaminoglycans and their Interactions with Proteins”;Gandhi N S and Mancera R L. in Chem. Biol. Drug Des. (2008), 72,455-482). There are two large groups (genera) of glycosaminoglycans:non-sulfated (such as hyaluronic acid) and sulfated (such as chondroitinsulfate, dermatan sulfate, keratan sulfate, heparin, and heparansulfate). Glycosaminoglycan chains are formed by disaccharide units ordisaccharides composed of an uronic acid (D-glucuronic or L-iduronic)and an amino sugar (D-galactosamine or D-glucosamine) such as thefollowing.

Formula 1: General structure of the disaccharide unit for the differenttypes of glycosaminoglycans.

Heparin is a polysaccharide of the glycosaminoglycan genus of compounds,formed by uronic acid (L-iduronic or D-glucuronic acid) andD-glucosamine, linked in alternating sequence. L-iduronic acid may be2-O-sulfated and D-glucosamine may be N-sulfated and/or 6-O-sulfated,and to a lesser extent N-acetylated or 3-O-sulfated (“Mapping andquantification of the major oligosaccharides component of heparin”,Linhardt R J, Rice K G, Kim Y S et al. in Biochem. J. (1988), 254,781-787). The major disaccharide repeating unit corresponds to thetrisulfated disaccharide, 2-O-sulfo-L-iduronic acid (1→4)2-N-sulfo-6-O-sulfo-D-glucosamine.

The origin of this structural variability present in heparinoligosaccharide chains is found in their biosynthesis and in themechanism regulating it. Thus, in the first stage of biosynthesis, atetrasaccharide fragment formed by glucose-galactose-galactose-xylose isbound to a protein core, starting the biosynthesis of the glycoproteinchain. Next, glucuronic acid (GlcA) residues and N-acetylglucosamine(GlcNAc) residues are alternatively incorporated forming apolysaccharide chain of approximately 300 units. At the same as thischain elongation occurs, and due to the intervention of various enzymes,modifications occur therein. Thus, the action ofN-deacetylase/N-sulfotransferase enzymes produce the N-deacetylation andN-sulfation of the GlcNAc units, turning them into N-sulfoglucosamine(GlcNS). A C5 epimerase catalyzes the transformation of certain units ofGlcA into iduronic acid (IdoA), followed by a 2-O-sulfation due toaction of a 2-O-sulfotransferase. Next, a 6-O-sulfotransferase,transfers a 6-O-sulfo group to GlcNS and GlcNAc units. Finally, a3-O-sulfotransferase acts on certain N-sulfo-6-O-sulfoglucosamine(GlcNS6S) units generating N-sulfo-3,6-di-O-sulfoglucosamine (GlcNS3S6S)residues.

The apparently random and incomplete nature of the initialN-deacetylation is what is mainly responsible for the introduction ofthe structural heterogeneity in heparin in the first phase of itsbiosynthesis. Structural variability with regard to the degree andpositions of sulfation is the result of the incomplete nature ofmodifications made by the biosynthetic enzymes that lead to theproduction of heparin sodium molecules with a variable disaccharidesubstitution pattern. Currently, no prior art NMR method norcorresponding composition for the highly accurate quantification ofindividual saccharides in GAG's and heparin molecules exist.

Heparin is preferably used as sodium salt, but it can also be used as asalt of other alkaline or alkaline-earth metals and is mainly used asantithrombotic and anticoagulant medicine (“Anticoagulant therapy formajor arterial and venous thromboembolism”, Tran HAM, Ginsberg J S inBasic principles and clinical practice (Colman R W, Marder V J, Clowes AW, George J N, Goldhaber S Z (Ed). Lippincott Williams and Wilkins;2006:1673-1688)).

Heparins can be classified depending on their molecular weight:unfractionated heparin (UFH), Low Molecular Weight Heparin (LMWH) with amean molecular weight lower than 8000 Da and Ultra Low Molecular WeightHeparin (ULMWH) with a mean molecular weight lower than 3000 Da(“Chemoenzymatic synthesis of homogenous ultra low molecular weightheparins”, Xu Y. et al. in Science (2011), 334, 498-501). LMWH and ULMWHcome from depolymerization of the original molecule of UFH, and itsmanufacturing process may introduce certain process-relatedcharacteristics in the molecule's structure. Thus, the resultingmolecule's structure derives on the one hand from the structure of theheparin used as starting material and on the other hand from thecharacteristic residues generated during preparation and characteristicmanufacturing method used.

The manufacturing process of enoxaparin sodium (β-elimination byalkaline treatment on benzyl ester of heparin in aqueous medium) andbemiparin sodium (β-elimination by alkaline treatment in non-aqueousmedium) generates as the majority species at the ends4,5-unsaturated-2-O-sulfo-uronic acid (ΔU2S), at the non-reducing end,and 2-N-sulfo-6-O-sulfoglucosamine, at the reducing end of the molecule.Additionally, the non-reducing end may have saccharides such as4,5-unsaturated-2-O-uronic acid (ΔU). At the reducing end of theaforementioned residue, it is possible to find2-N-sulfo-6-O-sulfomannosamine (the alkaline treatment catalyzes theepimerization in C2), in addition to another two species of 1,6-anhydroderivatives: 2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A) and2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M).

Formula 2: Structures present at the reducing and non-reducing end inenoxaparin and bemiparin sodium.

Residues are also generated in other low molecular weight heparinsaccording to their manufacturing process. For example, tinzaparinsodium, which is obtained by a method of β-elimination by treatment withheparinases, has at its non-reducing end 4,5-unsaturated-2-O-sulfouronicacid (ΔU2S).

Formula 3: Structures present at the reducing and non-reducing end intinzaparin sodium.

Dalteparin sodium is obtained by treatment with nitrous acid whichgenerates a 2,5-anhydro-mannitol residue at the reducing end of themolecule.

Formula 4: Structures present at the reducing and non-reducing end indalteparin sodium.

NMR spectroscopy allows for identification of the saccharide residuestypically present in heparin and low molecular weight heparin, such asthose formed during respective manufacturing processes.

One of the advantages associated with the use of NMR for structuralcharacterization is that, for its analysis, the samples do not requireprevious derivatization or chromatographic fractionation. In otherwords, the sample can be directly analysed by NMR, without the need forintermediate treatments.

NMR spectroscopy is used to determine the sequence of monosaccharideresidues present in these compounds and unequivocally determines theN-acetylation and N- and O-sulfation points throughout theoligosaccharide chain. Additionally, this technique allows specificallydetermining the orientation of the anomeric bonds and distinguishingbetween the iduronic acid of glucuronic acid epimers. (“AdvancingAnalytical Methods for Characterization of Anionic CarbohydrateBiopolymers”, Langeslay D. J. PhD Thesis UC Riverside 2013). However,given the high degree of microheterogeneity and polydispersity of thesecompounds, the complete characterization of heparins and low molecularweight heparins is currently still a challenge.

NMR can also be used to obtain information on those structural residuesassociated with the production process of heparins and of low molecularweight heparins, such as the state of epimerization of uronic acids(iduronic acid vs. glucuronic acid), ratio of sulfated and nonsulfated4,5-uronate residues at the non-reducing end (for low molecular weightheparins produced by a β-elimination method or treatment withheparinases).

Likewise, NMR can be used as a screening technique to determineimpurities present in glycosaminoglycans (“Analysis and characterizationof heparin impurities”, Beni S. et al. in Anal. Bioanal. Chem. (2011),399, 527-539).

Various NMR methods and experiments have been disclosed for thestructural characterization of glycosaminoglycans in general, andheparins and low molecular weight heparins in particular. Thus, forexample, ¹³C-NMR spectroscopy has been used to determine the degree ofsulfation in heparin sodium of different animal origin(“Characterization of Sulfation Patterns of Beef and Pig MucosalHeparins by Nuclear Magnetic Resonance”, Casu B. et al. inArzneim.-Forsch./Drug Res. (1996), 46, 472-477).

¹H-NMR spectroscopy has been the most widely used technique for thestudy of these compounds, since hydrogen is an abundant nucleus with ahigh gyromagnetic ratio. The region between 1.8-2.1 ppm comprises thesignals corresponding to the N-acetyl groups or methyl groups of thereducing ends which may be synthetically included. The region between2.8-4.6 ppm comprises the majority of the saccharide ring signals andhas a high degree of overlapping between them, which makes it difficultto extract structural information directly from this area.

Two-dimensional experiments (2D NMR) allow the shortcomings ofone-dimensional experiments to be overcome, i.e. shortcomings such asthe overlapping of signals. Two-dimensional spectra have two frequencydimensions and another signal intensity which allows them to become apowerful tool for assigning oligosaccharide structures derived fromheparin (“Characterization of currently marketed heparin products:composition analysis by 2D-NMR”, Keire D. A. et al. in Anal. Methods(2013), 5, 2984-2994).

TOCSY (TOtal Correlation SpectroscopY) spectroscopy can be used for thestructural analysis of oligosaccharides, since the information obtainedin that type of analysis allows the correlation of nuclei found in thesame spin system, in this case all the protons within the samemonosaccharide.

Another two-dimensional experiment of particular importance for thestructural characterization of this type of compounds is ¹H-¹³C HSQC(Heteronuclear Single-Quantum Correlation), which correlates ¹H protonchemical shifts with chemical shifts of ¹³C and permits assignment ofthe primary structures of oligosaccharides derived from GAGs(glycosaminoglycans) and the monosaccharide composition (“Structuralelucidation of the tetrasaccharide pool in enoxaparin sodium”, Ozug J.et al. in Anal. Bioanal. Chem. (2002), 403, 2733-2744; “Structuralfeatures of low molecular weight heparins affecting their affinity toantithrombin”, Bisio A. et al. in Thromb. Hemost. (2009), 102, 865-873).

The increase in spectral dispersion achieved with this two-dimensionaltechnique allows the quantification of the integrals of the signalswhich are superimposed in the corresponding one-dimensional spectra(“Low-molecular-weight heparins: structural differentiation bytwo-dimensional nuclear magnetic resonance spectroscopy”, Guerrini M. etal. Semin. Thromb. Hemost. (2007), 33, 478-487).

Nuclear magnetic resonance is a quantitative spectroscopy technique,since the intensity (amplitude) of the resonance lines is directlyproportional to the number of resonant nuclei (spin). This, inprinciple, makes it possible to precisely determine the quantity ofmolecular structures.

The increase in intensity of the magnetic fields used in NMR has allowedthe limits of detection to significantly be reduced. However, theabsence of precise methods that consider and control both theexperimental methods and the processing and evaluation of the spectrameans that measurements made on identical samples in variouslaboratories may significantly differ (“Validation of quantitative NMR”,Malz F. and Jancke H. in Journal of Pharmaceutical and BiomedicalAnalysis (2005), 38, 813-823).

The complexity of the nuclear magnetic resonance spectra ofglycosaminoglycans in general, and heparins and low molecular weightheparins and their derivatives in particular, has meant that to date nospecific validation methods have been developed which allowquantification of its characteristic signals, and therefore, thesuitable characterization and differentiation of these compounds. Inother words, prior art NMR methods have not successfully achieved highlyaccurate quantitation of each of the individual saccharide residuespresent GAG's and heparins.

With regard to GAG pharmaceutical products, their approval by healthauthorities of biosimilars and/or generics of certain low molecularweight heparins, as is the case of Enoxaparin sodium, requiresconfirmation of similarity between the biosimilar/generic and areference molecule. Similarity requires demonstration, among otheraspects, of a suitable degree of structural similarity between bothproducts. One of the basic aspects on a structural level that it isnecessary to demonstrate is that the relative proportion of themonosaccharides that form their oligosaccharide chains and afterstatistical evaluation, fulfil biosimilarity criteria. For them, themethod of the present invention is especially selective.

The present inventors have verified that, although the structuralcharacterization by nuclear magnetic resonance has been widely used forcharacterization of these compounds, the art provides no quantitativeanalysis methods of glycosaminoglycan analysis by means of NMR. Theabsence of these methods prevents the suitable comparability betweenidentical samples studied and assessed under not suitably establishedexperimental conditions.

Thus, it is possible to find in the literature publications wherein thevalues of relative proportion which are provided for the differentcomponent residues of these compounds significantly differ from oneanother, which clearly indicates that they are inadequate methods(“Generic versions of enoxaparin available for clinical use in Brazilare similar to the original drug”, Glauser B. F., Vairo B. C., OliveiraC. P. M., Cinelli L. P., Pereira M. S. and Mourao P. A. S. in J. Thromb.Haemost. (2011), 9, 1419-1422).

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome shortcomings ofprior NMR methods for analysis of GAG's. The present inventors havedeveloped a method which enables unequivocal differentiation of GAG'sfrom one another. As a result, the percentage values of monosaccharidecomposition provided by the present invention make it possible todifferentiate between low molecular weight GAG's obtained by differentmanufacturing processes and other molecular weight species of GAG's.

The present invention is useful for analysis of native forms of GAG's,modified forms of GAG's, as well as derivatives, free acid forms,freebase forms, and salt forms thereof. As used herein, the termglycosaminoglycan (GAG) is taken to mean at least the subgenera heparinsulfate, heparan sulfate, chondroitin sulfate, dermatan sulfate, keratansulfate, hyaluronic acid, others described herein, as well asderivatives, free acid forms, and salt forms thereof. Some of saidsubgenera are characterized as follows.

Hexuronic acid/ Linkage geometry between Name Hexose Hexosaminepredominant monomeric units Chondroitin GlcUA or GalNAc or GalNAc(4S) or′GlcUAβ1-3′GalNAcβ1-4 sulfate GlcUA(2S) GalNAc(6S) or GalNAc(4S,6S)Dermatan GlcUA or IdoUA or GalNAc or GalNAc(4S) or ′IdoUAβ1-3′GalNAcβ1-4sulfate IdoUA(2S) GalNAc(6S) or GalNAc(4S,6S) Keratan Gal or Gal(6S)GlcNAc or GlcNAc(6S) -Gal(6S)β1-4GlcNAc(6S)β1-3 sulfate Heparin GlcUA orGlcNAc or GlcNS or -IdoUA(2S)α1-4GlcNS(6S)α1-4 IdoUA(2S) GlcNAc(6S) orGlcNS(6S) Heparan GlcUA or IdoUA or GlcNAc or GlcNS or-GlcUAβ1-4GlcNAcα1-4 sulfate IdoUA(2S) GlcNAc(6S) or GlcNS(6S)Hyaluronan GlcUA GlcNAc -GlcUAβ1-3GlcNAcβ1-4

Suitable specific GAG's that can be employed in or characterized by theinvention are also selected from the group consisting of unfractionatedheparin (UFH), Low Molecular Weight Heparin (LMWH) with a mean molecularweight lower than 8000 Da and Ultra Low Molecular Weight Heparin (ULMWH)with a mean molecular weight lower than 3000 Da, enoxaparin, dalteparin,bemiparin, tinzaparin, all other known GAG's, all GAG's describedherein, as well as derivatives, free acid forms, freebase forms, andsalt forms thereof.

Suitable GAG's that can be employed in or characterized by the inventionare also selected from the group consisting of heparin, heparan,hyaluronan, keratan, dermatan, chondroitin, enoxaparin, bemiparin,dalteparin, tinzaparin, a salt of any of the preceding, a derivative ofany of the preceding, a sulfated form of any of the preceding, anon-sulfated form of any of the preceding, an ultra-low molecular weightform of any of the preceding, a low molecular weight form of any of thepreceding, a high molecular weight form of any of the preceding, anunfractionated form of any of the preceding, a fractionated form of anyof the preceding, and a combination of any two or more thereof.

The present invention provides a NMR method of determining the relativecontent of individual saccharide residues forming a respective GAG. Insome embodiments, the invention further comprises determining identityof individual saccharide residues forming a respective GAG. In someembodiments, the invention further provides a method of providing achemical shift pattern for a GAG.

The invention provides a method for quantification of the characteristicsignals of glycosaminoglycans in general (and heparins and low molecularweight heparins and their derivatives in particular) through the use ofone-dimensional nuclear magnetic resonance of ¹H-NMR and/ortwo-dimensional nuclear magnetic resonance of¹H-¹³C HSQC. The presentmethod employs quantification of characteristic ¹H and ¹³C chemicalshift signals for determination of the monosaccharide (saccharideresidue) content in oligosaccharide and polysaccharides of GAG's andheparins.

In the chemical shift range of 4.6-6.0 ppm for ¹H-NMR, the chemicalshift signals corresponding to the anomeric protons are found. Since itis an area much less populated with signals, it is possible to extract agreat deal of information from it. Furthermore, in the case of LMWHsobtained using a β-elimination mechanism, the range also contains thesignals corresponding to H4 of the non-reducing ends of the molecule.

In some embodiments, the hydrogen of the saccharide being quantified isselected from the group consisting of the H1 hydrogen, H2 hydrogen, H3hydrogen, H4 hydrogen, H5 hydrogen, and H6 hydrogen of the correspondingC1, C2, C3, C4, C5, and C6 carbons of the saccharide. In someembodiments, the chemical shift signal range for the hydrogen beingquantified is in the range of 3.2 to 6 ppm, 4.6-6.0 ppm, or 3.2 to lessthan 4.6 ppm.

In some embodiments, the carbon of the saccharide being quantified isselected from the group consisting of the C1 carbon, C2 carbon, C3carbon, C4 carbon, C5 carbon and C6 carbon of the saccharide. In someembodiments, the chemical shift signal range for the carbon beingquantified is in the range of about 55 to about 115 ppm or about 25-25ppm.

An aspect of the invention provides a ¹H-NMR one-dimensional nuclearmagnetic resonance and/or ¹H-¹³C HSQC two-dimensional nuclear magneticresonance method for the analysis of a composition comprising saccharideresidues present in, or derived from, GAG, the method comprising thesteps of:

-   -   a) providing a composition comprising DMMA (dimethyl malonic        acid or deuterated derivative thereof), at least one deuterated        solvent, and at least one glycosaminoglycan comprising        saccharide residues comprising respective anomeric hydrogens;    -   b) conducting at least one NMR analysis on said composition,        wherein said at least one NMR analysis is selected from the        group consisting of ¹H-NMR one-dimensional analysis and ¹H-¹³C        HSQC two-dimensional analysis, and wherein said DMMA is employed        as an internal reference for concentration dependent response of        chemical shift signal intensity for ¹H and/or ¹³C, thereby        providing at least one respective spectrum (numerical and/or        graphical spectrum) comprising respective chemical shift signals        for said respective anomeric hydrogens;    -   c) normalizing said respective chemical shift signals for said        respective anomeric hydrogens with respect to the ¹H and/or ¹³C        chemical shift signal of said DMMA;    -   d) correlating said respective chemical shift signals for        respective anomeric hydrogens to one or more reference chemical        shift signals for respective anomeric hydrogens of reference        saccharides selected from the group consisting of:        4,5-unsaturated 2-O-sulfo-uronic acid (ΔU2S), 4,5-unsaturated        uronic acid (ΔU), 2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A),        2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M),        2-N-sulfo-6-O-sulfoglucosamine (ANS6S), 2,5-anhydro mannitol,        N-sulfoglucosamine, glucuronic acid,        N-sulfo-6-O-sulfoglucosamine, 2-O-sulfoiduronic acid, iduronic        acid, N-sulfo-3-O-sulfoglucosamine,        N-sulfo-3,6-di-O-sulfoglucosamine, galacturonic acid, xylose,        N-acetylglucosamine and N-acetyl-6-O-sulfoglucosamine, thereby        providing the identity and relative proportion of individual        saccharide residues present in said GAG.

The invention also provides a nuclear magnetic resonance (NMR) methodfor quantifying content of saccharide in one or more glycosaminoglycansin a composition, the method comprising at least the step of:

-   -   providing a composition comprising: i) at least one        glycosaminoglycan (GAG) comprising plural saccharides comprising        respective anomeric or target hydrogen atoms exhibiting        respective anomeric or target ¹H chemical shift signals when        analyzed by NMR and further comprising respective anomeric or        target carbon atoms covalently bound to said anomeric or target        hydrogens atoms and exhibiting respective anomeric or target ¹³C        chemical shift signals when analyzed by NMR; ii) at least one        reference compound comprising a hydrogen atom having a NMR        signal t1 longitudinal relaxation time of 1 s or less and a        respective carbon atom covalently bound to said hydrogen atom,        wherein the at least one reference compound exhibits a reference        ¹H NMR chemical shift signal separated from said anomeric or        target ¹H chemical shift signals and further exhibits a        reference ¹³C NMR chemical shift signal separated from said        anomeric or target ¹³C chemical shift signals, and said        reference ¹H chemical shift signal and said reference ¹³C        chemical shift signal each exhibits a concentration dependent        intensity; and iii) at least one deuterated solvent for said        glycosaminoglycan and said at least one reference compound.

In some embodiments, the method further comprises comparing the at leastone respective spectrum for a sample GAG to at least one referencespectrum for a reference GAG, said spectra having been obtained undersubstantially the same conditions.

The method can further comprise: a) comparing the intensities of saidanomeric or target ¹H chemical shift signals to the intensity of thereference ¹H chemical shift signal; b) comparing the intensities of saidanomeric or target ¹³C chemical shift signals to the intensity of thereference ¹³C chemical shift signal; or c) a combination of a) and b).

The method can further comprise: a) obtaining the integrals for saidanomeric or target ¹H chemical shift signals and said reference ¹Hchemical shift signal; b) obtaining the integrals for said anomeric ortarget ¹³C chemical shift signals and said reference ¹³C chemical shiftsignal; or c) a combination of a) and b).

The method can further comprise: a) dividing said integrals for saidanomeric or target ¹H chemical shift signals by the integral of saidreference ¹H chemical shift signal to provide normalized values of saidintegrals for said anomeric or target ¹H chemical shift signals; b)dividing said integrals for said anomeric or target ¹³C chemical shiftsignals by the integral of said reference ¹³C chemical shift signal toprovide normalized values of said integrals for said anomeric or target¹³C chemical shift signals; or c) a combination of a) and b).

As used herein, the term “anomeric carbon” refers to a carbon atomhaving an anomeric hydrogen covalently bound thereto.

The method can further comprise: a) determining the relative proportionsof said anomeric or target hydrogens with respect to the total amount ofanomeric or target hydrogens present in said glycosaminoglycan toprovide the content of saccharide in said one or moreglycosaminoglycans; b) determining the relative proportions of saidanomeric or target carbons with respect to the total amount of anomericor target carbons present in said glycosaminoglycan to provide thecontent of saccharide in said one or more glycosaminoglycans; or c) acombination of a) or b).

The method can further comprise: a) correlating the relative proportionsof said anomeric or target hydrogens with the relative proportions ofcorresponding saccharides present in said glycosaminoglycan; b)correlating the relative proportions of said anomeric or target carbonswith the relative proportions of corresponding saccharides present insaid glycosaminoglycan; or c) a combination of a) or b).

The method can further comprise: a) developing a calibration curve forsaid at least one glycosaminoglycan; b) developing a calibration curvefor said at least one reference compound; c) developing calibrationcurves for said plural saccharides; or d) a combination of any two ormore of the above.

The method can further comprise: providing a reference ¹H NMR spectrumand/or reference ¹³C NMR spectrum for each of said plural saccharidesbeing quantified.

The method can further comprise: providing a reference ¹H-NMR spectrumand/or reference ¹³C-NMR spectrum for said at least oneglycosaminoglycan.

The method can further comprise: a) determining the relative proportionsof anomeric or target hydrogens in one or more referenceglycosaminoglycans; b) determining the relative proportions of anomericor target carbons in one or more reference glycosaminoglycans; or c) acombination of a) or b).

The method can further comprise: a) correlating said relativeproportions of anomeric or target hydrogens with the relativeproportions of corresponding saccharides present in said one or morereference glycosaminoglycans; b) correlating said relative proportionsof anomeric or target carbons with the relative proportions ofcorresponding saccharides present in said one or more referenceglycosaminoglycans; or c) a combination of a) or b).

The suitable quantification of the characteristic signals of theseproducts and of their monosaccharide composition or characteristicresidues, allows for differentiation of polysaccharides from oneanother, and in the case of the low molecular weight heparins, toconfirm that the products have been manufactured according to thedeclared method.

This quantification of the residues is obtained in percentage values andin relative form to the complete structure of each heparin therebyproviding a characteristic chemical shift spectrum (NMR fingerprint) ofeach one of the structures and allowing for an unequivocal deduction andidentification both of the GAG analyzed and of the process by which itwas prepared. Accordingly, the NMR method of present invention can beused as both a quality control system (i.e. a method to determinewhether the GAG analyzed corresponds to a reference GAG or has beenadulterated) and/or as a method for the identification of new GAG's.

With the aim of establishing a selective quantitative method todetermine the proportion of the signals corresponding to these residuespresent in the structure of these compounds, which allows the suitablecomparison between results and which makes it possible to avoid theerror associated to the conditions in which NMR experiments areperformed, the inventors have found that the use of dimethylmalonic acid(DMMA) as internal standard in ¹H-NMR and ¹HSQC analyses for therelative quantification of the signals corresponding to the differentcomponent monosaccharides is suitable, since among other aspects it hasa longitudinal relaxation time (T1) of under 1 second similar to the T1of the anomeric protons and carbons (“Molecular Weight of Heparin using13C Nuclear Magnetic Resonance Spectroscopy”, Desai U. R. and LinhardtR. J., in J. Pharm. Sci. (1995), 84(2), 212-215), which allows a goodtransfer of polarization and, therefore, an increase in intensity of thesignals, which makes it suitable for this purpose.

Thus, in some embodiments, the instant method for NMR analysis (¹H-NMRand/or ¹H-¹³C HSQC), of GAG's employs DMMA as internal standard for thequantification of the characteristic signals of said GAG's.Surprisingly, it has been found that the selection DMMA allows forquantitative determination, by percentage means or relative proportion,of the characteristic signals in glycosaminoglycans, in particular thosetypically related to heparins. The method provides high specificity,high accuracy, high repeatability (reproducibility) and high linearity(in a certain concentration range for DMMA) between the chemical shiftsignal intensity and the concentration of the characteristic residues ofthe target GAG's, thereby enabling development of a quantitativeanalytical method for said GAG's. The use of DMMA is particularlypreferred because of the combination of at least the followingproperties: a) solubility in solvent(s) in which the GAG is soluble; b)a ¹H reference chemical shift signal separated from the target ¹Hchemical shift signals of the GAG; c) a ¹³C reference chemical shiftsignal separated from the target ¹³C chemical shift signals of the GAG;d) a linear relationship between concentration of DMMA and referencechemical shift signal(s) intensity at a concentration that is acceptablefor GAG-containing samples; e) substantial independence of said linearrelationship upon presence or absence of GAG; and f) lack of degradationof GAG by DMMA during NMR analysis under experimental conditionsemployed.

In some aspects, the invention provides a nuclear magnetic resonance(NMR) method for quantifying the content of saccharide(s) in one or moreglycosaminoglycans in a composition, the method comprising at least thestep of:

-   -   providing a composition comprising: i) at least one        glycosaminoglycan (GAG) comprising plural saccharides comprising        respective anomeric hydrogen atoms exhibiting respective        anomeric ¹H and/or ¹³C chemical shift signals when analyzed by        NMR; ii) at least one reference compound comprising a hydrogen        atom having a NMR signal T1 (longitudinal relaxation time) of        about 1 s or less, wherein the at least one reference compound        exhibits a reference ¹H-NMR chemical shift signal separated from        said anomeric ¹H chemical shift signals, and said reference ¹H        chemical shift signal exhibits a concentration dependent        intensity (or amplitude); and iii) at least one deuterated        solvent for said glycosaminoglycan and said at least one        reference compound.

Embodiments of the invention provide a composition comprising: a) atleast one glycosaminoglycan (GAG) comprising plural saccharidescomprising respective target hydrogen atoms and respective carbon atomsexhibiting respective target ¹H and/or ¹³C chemical shift signals whenanalyzed by NMR; b) at least one reference compound comprising ahydrogen atom having a NMR signal t1 longitudinal relaxation time ofabout 1 s or less, wherein the at least one reference compound exhibitsa reference ¹H-NMR chemical shift signal separated from said target ¹Hchemical shift signals, and said reference ¹H chemical shift signalexhibits a concentration dependent intensity (or amplitude); and iii) atleast one deuterated solvent for said glycosaminoglycan and said atleast one reference compound.

Embodiments of the invention provide a composition comprising: a) atleast one GAG present at a concentration of about 0.005 to about 1mg/μL, about 0.01 mg/μL to about 0.5 mg/μL, or about 0.02 to about 0.2mg/μL; b) DMMA present at a concentration of about 0.05 to about 5 mM,about 0.1 to about 5 mM, about 0.1 to about 4 mM, about 0.1 to about 3mM, or about 0.2 to about 2.5 mM; and c) at least one deuteratedsolvent.

The method can further comprise conducting at least one NMR analysis, onsaid composition, selected from the group consisting of one-dimensional(1D) ¹H-NMR analysis, two-dimensional (2D) ¹H-¹³C-NMR analysis, or acombination of said analyses, to provide at least one GAG NMR spectrum.Said at least one NMR analysis comprises exposing said composition toplural magnetic pulses such that the time between individual magneticpulses (d1) is about the T1 or more of the target H being analyzed, oris about one second or more, or about 1 s to about 10 s, or about 1 s toabout 5 s, or about 1 s to about 2 s. In some embodiments, the target His an anomeric hydrogen atom of a saccharide residue.

The method can further comprise comparing the intensities of saidanomeric or target ¹H chemical shift signals to the intensity of thereference ¹H chemical shift signal.

The method can further comprise comparing the intensities of saidanomeric or target ¹³C chemical shift signals to the intensity of thereference ¹³C chemical shift signal.

The method can further comprise normalizing the intensities of saidanomeric or target ¹H chemical shift signals relative to the intensityof the reference ¹H chemical shift signal.

The method can further comprise normalizing the intensities of saidanomeric or target ¹³C chemical shift signals relative to the intensityof the reference ¹³C chemical shift signal.

The method can further comprise obtaining the integrals for saidanomeric or target ¹H chemical shift signals and said reference ¹Hchemical shift signal. Said integrals for said anomeric or target ¹Hchemical shift signals can be divided by the integral of said reference¹H chemical shift signal to provide normalized values of said integralsfor said anomeric or target ¹H chemical shift signals. The method canfurther comprise determining the relative proportions of said anomericor target hydrogens with respect to the total amount of anomeric ortarget hydrogens present in said glycosaminoglycan to provide thecontent of saccharide in said one or more glycosaminoglycans.

The method can further comprise correlating the relative proportions ofsaid anomeric or target hydrogens with the relative proportions ofcorresponding saccharides present in said glycosaminoglycan.

The method can further comprise: a) developing a calibration curve forsaid at least one glycosaminoglycan; b) developing a calibration curvefor said at least one reference compound; c) developing calibrationcurves for said plural saccharides; or d) a combination of any two ormore of the above.

The method can further comprise providing a reference ¹H-NMR spectrumand/or reference ¹³C-NMR spectrum for each of said plurals saccharidesbeing quantified

The method can further comprise determining the relative proportions ofanomeric or target hydrogens in one or more referenceglycosaminoglycans. The method can further comprise correlating saidrelative proportions of anomeric or target hydrogens with the relativeproportions of corresponding saccharides present in said one or morereference glycosaminoglycans.

The method can further comprise embodiments, wherein said concentrationdependent intensity is substantially linear in the range of about 0.2 mMto about 2.5 mM of said at least one reference compound. The linearitycan have a correlation coefficient (R²) of ≥0.90, ≥0.95, ≥0.98, or≥0.99.

The method can further comprise using the singlet ¹H-NMR chemical shiftof 3-(trimethylsilyl)-priopionic-D4 acid as a chemical shift referencefor 0 ppm.

The method can further comprise embodiments, wherein the two-dimensional¹H-¹³C-NMR analysis is heteronuclear single-quantum correlation (HSQC).

Other aspects of the invention provide a composition comprising:

-   1) at least one glycosaminoglycan comprising at least one saccharide    comprising anomeric or target hydrogen exhibiting respective    anomeric or target ¹H chemical shift signal in the range of about    4.6 to about 6 ppm or about 3.2 to about 6 ppm when analyzed by NMR;-   2) at least one reference compound comprising at least one reference    hydrogen atom having a NMR signal T1 (longitudinal relaxation time)    of about 1 s or less, wherein the at least one reference hydrogen    exhibits a reference ¹H NMR chemical shift signal separated from    said anomeric or target ¹H chemical shift signals, and said    reference ¹H chemical shift signal exhibits a concentration    dependent signal intensity; and-   3) at least one deuterated solvent for said glycosaminoglycan and    said at least one reference compound.

The invention also provides embodiments wherein said glycosaminoglycanis an oligosaccharide or polysaccharide.

The invention also provides embodiments wherein the deuterated solventis selected from the group consisting of any deuterated solvent in whichthe GAG is soluble at the concentration range described herein, anycombination of deuterated solvents in which the GAG is soluble at theconcentration range described herein, D₂O, CD₃CO₂D, CD₃OD, CCl₃OD,(CD₃)₂SO, CD₃CN, (CD₃)₂NC(O)D, and a combination of D₂O and at least oneother deuterated solvent. In some embodiments, said at least one otherdeuterated solvent is selected from the group consisting of CD₃CO₂D,CD₃OD, CCl₃OD, (CD₃)₂SO, CD₃CN, and (CD₃)₂NC(O)D. Deuterated mineralacid and/or alkali can be used to adjust the pH of the composition asneeded.

The invention also provides embodiments wherein the at least onesaccharide is selected from the group consisting of 4,5-unsaturated 2-Osulfo-uronic acid (ΔU2S), 4,5-unsaturated uronic acid (ΔU),2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A),2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M),2-N-sulfo-6-O-sulfoglucosamine (ANS6S), 2,5-anhydro mannitol,N-sulfoglucosamine, glucuronic acid, N-sulfo-6-O-sulfoglucosamine,2-O-sulfoiduronic acid, iduronic acid, N-sulfo-3-O-sulfoglucosamine,N-sulfo-3,6-di-O-sulfoglucosamine, galacturonic acid, Xylose,N-acetylglucosamine and N-acetyl-6-O-sulfoglucosamine.

The invention also provides embodiments wherein said at least onereference compound is present at a concentration in the range of about0.2 mM to about 2.5 mM.

The invention also provides embodiments wherein said reference ¹H-NMRchemical shift signal is outside the range of about 4.6 to about 6.0 ppmor outside the range of about 3.2 to about 6 ppm.

The invention also provides embodiments wherein said at least oneinternal reference compound exhibits a reference ¹H-NMR chemical shiftsignal within the range of about 1.2 ppm to about 1.7 ppm, about 1.2 ppmto about 1.6 ppm, about 1.2 to about 1.5 ppm, about 1.3 to about 1.5ppm, about 1.4 to about 1.5 ppm, about 1.3 ppm, and about 1.4 ppm, eachsaid range and value being inclusive of the range limits especially asdefined by the definition of the term “about” as used herein, and eachsaid chemical shift being relative to TSP as defining 0 ppm. In someembodiments, said reference ¹H-NMR chemical shift signal is a singlet.

In some embodiments, said at least one internal reference compoundexhibits a reference ¹H-NMR chemical shift signal of greater than 0 ppmand less than about 1.8 ppm, within the range of about 2.2 to about 3ppm, or within the range of about 6.2 to about 7.2 ppm, each said rangeand value being inclusive of the range limits especially as defined bythe definition of the term “about” as used herein, and each saidchemical shift being relative to TSP as defining 0 ppm.

The invention also provides embodiments wherein said at least oneinternal reference compound exhibits a ¹³C-NMR chemical shift signalwithin the range of about 25 to about 27 ppm or within the range ofabout 26 ppm to about 27 ppm, inclusive of the range limits especiallyas defined by the definition of the term “about” as used herein, andeach said chemical shift being relative to TSP as defining 0 ppm. Insome embodiments, said reference ¹³C-NMR chemical shift signal is asinglet. In some embodiments, the reference ¹³C-NMR chemical shiftsignal is in the range of greater than 0 pp to less than 22 ppm, or 25ppm to less than 55 ppm.

The invention also provides embodiments wherein said reference ¹H-NMRchemical shift is relative to the singlet chemical shift of3-(trimethylsilyl)-priopionic-D4 acid assigned as 0 ppm.

The invention also provides embodiments wherein said at least onereference compound is dimethylmalonic acid (DMMA), derivative thereof,salt thereof or combination of any two or more thereof.

The invention also provides embodiments wherein said at least onereference compound is present at a known or predetermined concentrationor amount.

The invention also provides embodiments wherein said at least oneglycosaminoglycan is present at a known or predetermined concentrationor amount.

The invention also provides embodiments wherein said at least oneglycosaminoglycan is selected from the group consisting of heparin,heparan, enoxaparin, bemiparin, dalteparin, tinzaparin, a salt of any ofthe preceding, a derivative of any of the preceding, or a combinationthereof. The method and composition of the invention are suitable foruse in quantifying sulfated and/or non-sulfated saccharides present inglycosaminoglycans.

Another aspect of the invention provides a software program forconducting one or more of the methods disclosed herein. Another aspectof the invention provides a NMR spectrometer controlled by said softwareor with said software installed therein. Another aspect of the inventionprovides computer with said software installed therein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Diagram of the biosynthetic process of heparin.

FIG. 2: Specificity. ¹H-NMR spectra: a) SEL-I-D2O: spectrum of D₂Osolvent; b) SEL-II-D2O-TSP: spectrum of combination of D₂O solvent andTSP; c) SEL-III-DMMA-D2O: spectrum of combination of D₂O solvent andDMMA; d) SEL-IV-ENOX-DMMA-D2O: spectrum of combination of enoxaparin,D₂O solvent and DMMA.

FIG. 3: Specificity. ¹H-¹³C HSQC spectra of SEL-IV-ENOX-DMMA-D2O. Thespectrum was obtained under the following conditions: Temperature: 298.0K; number of scans: 12; receiver gain: 2050.0; relaxation delay: 1.8;pulse width: 11.11; acquisition time: 0.1068 s; JCH: 170; spectrometerfrequency: 800.13, 201.49; spectral width (ppm): 4795.4, 24154.6; lowestfrequency: 447.9, 575.7.

FIG. 4: ¹H-NMR linearity. Graph depicting the linearity of concentration(mM) of DMMA (variable X) versus the integral (variable Y) of its ¹Hchemical shift signal. Under the conditions of the assay employed, thelinearity is defined by the following equation:y=1,136,561.5669x−42,152.0764 with an R² value of 0.9985.

FIG. 5: ¹H-¹³C HSQC linearity. Graph depicting the linearity ofconcentration (mM) of DMMA (variable X) versus the integral (variable Y)of its ¹H chemical shift signal. Under the conditions of the assayemployed, the linearity is defined by the following equation:y=59,069,197.0868x−4,422,535.0841, with an R² value of 0.9966

FIG. 6: ¹H-NMR spectrum of enoxaparin sodium. The spectrum was obtainedunder the following conditions: Temperature: 298.0 K; number of scans:12; receiver gain: 2.0; relaxation delay: 13.5; pulse width: 10.69;acquisition time: 6.8157 s; JCH: 1; spectrometer frequency: 800.13;spectral width (ppm): 12.0.

FIG. 7: ¹H-¹³C HSQC spectrum of enoxaparin sodium. The spectrum wasobtained under the following conditions: Temperature: 298.0 K; number ofscans: 12; receiver gain: 2050.0; relaxation delay: 1.8; pulse width:10.69; acquisition time: 0.1068 s; JCH: 170; spectrometer frequency:800.13, 201.19; spectral width: 4795.4, 24154.6; lowest frequency:447.9, 575.7.

ABBREVIATIONS AND ACRONYMS

The following abbreviations and acronyms are used in the presentspecification:

-   NMR: Nuclear magnetic resonance-   HSQC: Heteronuclear Single-Quantum Correlation-   GAG: Glycosaminoglycan-   UFH: Unfractionated heparin-   LMWH: Low Molecular Weight Heparin-   ULMWH: Ultra Low Molecular Weight Heparin-   Da: Dalton-   ΔU2S: 4,5-unsaturated 2-O sulfo uronic acid-   ΔU: 4,5-unsaturated uronic acid-   1,6-an. A: 2-N-sulfo-1,6-anhydroglucosamine-   1,6-an.M: 2-N-sulfo-1,6-anhydro-mannosamine-   TOCSY: TOtal Correlation SpectroscopY-   TSP: Sodium salt of 3-(Trimethylsilyl)-Propionic-D4 acid-   DMMA: Dimethylmalonic acid, derivative thereof, and or salt thereof-   MHz: Megahertz-   ppm: parts per million-   δ: chemical shift-   SW: Spectral width-   TD: Time domain-   T1: Longitudinal relaxation time-   ANS: N-sulfoglucosamine-   G: Glucuronic acid-   ANS6S: N-sulfo-6-O-sulfoglucosamine-   I2S: 2-O-sulfoiduronic acid-   I: duronic acid-   ANS3S: N-sulfo-3-O-sulfoglucosamine-   Gal: Galacturonic acid-   Xyl: Xylose-   ANAc: N-acetylglucosamine-   A6S: 6-O-sulfoglucosamine-   A6OH: Glucosamine-   G2S: Sulfoglucuronic 2-O acid-   M: Mannosamine-   MNS6S: N-sulfo-6-O-sulfomannosamine-   Epox: Epoxide-   αred: α anomer-   βred: β anomer.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “monosaccharide residue present in heparin chains”refers to all monosaccharide residues or components that are typicallypresent in LMWH/UFH/GAG chains. These residues are generally selectedfrom the group consisting of 4,5-unsaturated 2-O sulfo uronic acid(ΔU2S), 4,5-unsaturated uronic acid (ΔU),2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A),2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M),2-N-sulfo-6-O-sulfoglucosamine (ANS6S), 2,5-anhydro mannitol,N-sulfoglucosamine, glucuronic acid, N-sulfo-6-O-sulfoglucosamine,2-O-sulfoiduronic acid, iduronic acid, N-sulfo-3-O-sulfoglucosamine,N-sulfo-3,6-di-O-sulfoglucosamine, galacturonic acid, Xylose,N-acetylglucosamine and N-acetyl-6-O-sulfoglucosamine.

The invention provides a composition for NMR analysis of GAG's. Thecomposition comprises at least one GAG (comprising plural saccharideresidues having respective anomeric or target hydrogens) and at leastone internal reference compound having a T1 for hydrogen of about the T1for the anomeric or target hydrogen of at least one said pluralsaccharide residues. The composition can further comprise a deuteratedsolvent in which said at least one GAG is at least partially soluble oris fully soluble and in which said at least one internal referencecompound is at least partially soluble or is fully soluble.

In some embodiments, the composition comprises at least one GAG, DMMA,and at least one deuterated solvent. The content of said components inthe composition can be as described herein. The composition can be asample composition.

A GAG comprises plural saccharide residues or at least two differentsaccharide residues. A saccharide residue will usually comprise ananomeric and/or target hydrogen atom(s). Anomeric or target hydrogenstypically exhibit a T1 of about 1 sec or 1±0.2 sec or about 1 sec orless, of ≤ about 1 sec. Anomeric hydrogens typically exhibit a chemicalshift signal of about 4.6-6 ppm for ¹H-NMR. At least hydrogen atom(s) ofthe internal reference standard will exhibit a singlet chemical shiftreference signal outside the range of about 4.6-6 ppm, and the intensityof said reference signal will be concentration dependent. At leasthydrogen atom(s) of the internal reference standard can exhibit asinglet chemical shift reference signal in the range of about 1.2 toabout 1.4 ppm for ¹H-NMR analysis and a singlet chemical shift signalwithin the range of about 26 ppm to about 27 ppm ¹³C-NMR. Theconcentration versus intensity typically exhibits an approximatelylinear relationship when said reference standard is present in thecomposition at least in the concentration range of about 0.2 mM to about2.5 mM, and the linearity of said relationship can also exist outsidesaid concentration range. The linearity can have a correlationcoefficient of ≥0.90, ≥0.95, ≥0.98, or ≥0.99.3-(trimethylsilyl)-priopionic-D4 acid (deuterated TSP) can be used as aninternal chemical shift signal reference for 0 ppm in ¹H-NMR. Theconcentration of total amount (concentration) of GAG, or at least one ofits saccharide residues, is typically in the range of about 15 to about700 mM, about 25 to about 600 mM, about 30 to about 500 mM, about 30 toabout 400 mM, about 33 to about 333 mM, about 0.005 to about 1 mg/μL,about 0.01 mg/μL to about 0.5 mg/μL, or about 0.02 to about 0.2 mg/μL.At least one hydrogen of the internal reference standard will exhibit aT1 that approximates, is equal to, or is less than the T1 of theanomeric or target hydrogen of said saccharide residue(s), e.g. a T1 ofabout 1 sec or 1±0.2 sec or about 1 sec or less, of ≤about 1 sec.Combinations of two or more deuterated solvents can be used. An aqueousdeuterated solvent can be used, e.g. D₂O or D₂O in combination with oneor more deuterated organic solvents. The solvent or combination ofsolvents used will dissolve at least a portion of the GAG or willdissolve all of the GAG in the composition. The free acid or salt formof DMMA can be used.

The present invention provides a NMR method of determining the identityand relative content of individual saccharide residues forming arespective GAG. A NMR method of the invention is generally performed by:a) providing a sample composition comprising at least one GAG(comprising plural saccharide residues, each comprising at least onerespective hydrogen and at least one respective carbon), at least oneinternal reference compound (as described herein for concentrationstandard), TSP (internal standard defining 0 ppm), and at least onedeuterated solvent; b) conducting a 1D or 2D NMR analysis on said samplecomposition to obtain at least one reference chemical shift signal (forat least one hydrogen of said at least one internal reference and/or forat least one carbon of said at least one internal reference) and toobtain plural saccharide residue chemical shift signals (for said atleast one respective hydrogens and/or for said at least one respectivecarbon of said respective said plural saccharide residue); c)normalizing said plural saccharide residue chemical shift signalsrelative to (with respect to) said at least one reference chemical shiftsignal(s); d) determining the relative percentage of individual ones ofsaid plural saccharide residues in said at least one GAG; and e)correlating said plural saccharide residue chemical shift signals toknown chemical shift signals for saccharide residue standards todetermine the identity of individual ones of said plural saccharideresidue. The above steps after b) need not be conducted sequentially,meaning steps c) through e) can be conducted in any order.

The relative percentage of individual saccharide residues in a GAG canbe determined according to the following formula:

${\%\mspace{14mu}{signal}\mspace{14mu} X} = {\frac{{normalized}\mspace{14mu}{value}\mspace{14mu}{for}\mspace{14mu}{signal}\mspace{14mu} X}{\Sigma\mspace{14mu}{normalized}\mspace{14mu}{value}\mspace{14mu}{for}\mspace{14mu}{all}\mspace{14mu}{the}\mspace{14mu}{signals}} \times 100.}$wherein:X is the target hydrogen.normalized value for signal X is the chemical shift signal intensity forX after said signal has been normalized relative to the referencechemical shift signal, and Σ normalized value for all the signals is thesum total of all the normalized values.

A chemical shift signal pattern (or “NMR fingerprint”), as used herein,refers to the set of the signals corresponding to the peaks found in adetermined NMR spectrum, whether one-dimensional ¹H-NMR and/ortwo-dimensional ¹H-¹³C HSQC, or the absence thereof, in the relativeproportions of its normalized integrals indicated by the parameter“relative proportion (%)”. The chemical shift signal pattern can be agraphical spectrum (visual linear graph) or a numerical spectrum (groupof values in a data set). The absence of particular chemical shiftsignals corresponds to the absence of respective saccharide residues andthe presence of particular chemical shift signals corresponds to thepresence of respective saccharide residues. The chemical shift signalpattern is used to identify, characterize, specify the saccharidecomposition of a corresponding GAG.

The method and composition of the invention can be practiced with anyGAG known to date and any as yet unknown GAG, meaning any GAG discoveredor first prepared after the date of the present invention.

Experimental Assays

The quantitative NMR assays have been performed using a Bruker AVIII-600o AVIII-800 nuclear magnetic resonance (NMR) spectrometer. Reagents usedwere deuterium oxide (D₂O) 99.9%, sodium salt of3-(Trimethylsilyl)-Propionic-D4 acid (TSP) and Dimethylmalonic acid(DMMA, standard for quantitative NMR, TraceCERT grade) as internalstandard.

-   a) Equipment Conditions    -   Frequency: 1H: 600/800 MHz, 13C: 150,9/201.2 MHz    -   Temperature: 298 K-   b) Acquisition Parameters (quantitative ¹H NMR)    -   90° pulse: it is determined from a qualitative 1H spectrum    -   Acquisition window: SW=10-12 ppm/TD=64-128 k    -   Inter scans delay d1 must fulfil the condition d1+AQ≥20 s    -   No. of scans: 12-   c) Acquisition Parameters (HSQC)    -   90° pulse: it is determined from a qualitative ¹H spectrum    -   Acquisition window: SW2 (¹H)=6 ppm/TD(F2)=1 k        -   SW1 (¹³C)=120 ppm/TD(F1)=256−384    -   Time between pulses d1=1.8−2 s    -   No. of scans: 12-   d) Processing Parameters (¹H)    -   Processing window: SI=64−256 K    -   Window function: None    -   Phase adjustment: manual    -   Baseline adjustment: automatic (abs)-   e) Processing Parameters (HSQC)    -   Processing window: SI(F2)=2 k    -   Processing function: QSINE, SSB=2    -   Phase adjustment: manual    -   Baseline adjustment: automatic-   f) Preparation of the Sample: the Following Solutions were Prepared:    -   Solution A (TSP) 1 mg/mL    -   Solution B (D₂O-TSP) 0.002 mg/mL: 40 μL A (TSP)+19.96 mL D₂O,        Total volume=20 mL    -   Solution C (DMMA) 1.2 mg dimethylmalonic/mL of D₂O    -   Test sample: 50 mg of product to study in 500 μL of the solution        B (D₂O-TSP) and add 100 μL of solution C (DMMA) and place in 5        mm-diameter NMR tube.

The NMR tube containing the sample is introduced in the spectrometer.Then, the homogeneity of the magnetic field is adjusted and the harmonyof the wave is optimized for the ¹H and ¹³C nuclei. A qualitative ¹Hspectrum is then performed, with parameters similar to theaforementioned, except the following:

-   -   Time between pulses d1=1−2 s; No. of scans: 1-4.

Then, the value of the 90° pulse is determined with automatic pulseprogram (TOPSPIN). Next, the quantitative ¹H spectrum is performed, withthe parameters indicated in the analytical method and the 90° pulsevalue (P1) previously determined. After the HSQC spectrum is obtainedwith the aforementioned parameters. The spectra obtained are thenprocessed according to the aforementioned parameters, taking as chemicalshift reference, the TSP-d4 signal at 0 ppm.

The dimethylmalonic acid reference chemical shift signals appear at thefollowing approximate chemical shifts:

-   -   ¹H NMR: singlet that appears at about 1.2 to about 1.5 ppm,        about 1.3 to about 1.5 ppm, about 1.4 to about 1.5 ppm, about        1.3 ppm, and about 1.4 ppm;    -   HSQC: signal at about 1.2 to about 1.5 ppm, about 1.3 to about        1.5 ppm, about 1.4 to about 1.5 ppm, about 1.3 ppm, and about        1.4 ppm (¹H) and about 25 to about 27 ppm, about 26 ppm to about        27 ppm (¹³C).

It should be understood that all chemical shift signals described hereinare approximate and can vary slightly according to experimentalconditions; however, said signals are obtained within the specifiedranges when corresponding NMR analyses are conducted as describedherein. Moreover, all ranges specified herein are inclusive of the rangelimits and all integer and fractional values therein especially asdefined by the definition of the term “about” as used herein, and eachsaid chemical shift is relative to TSP as the internal referencedefining 0 ppm.

The parameters assessed to determine validation of the method forquantitative NMR have been the following:

Specificity

This aspect of the method determines the capacity of the analyticalmethod for measuring and/or identifying, simultaneously or separately,the analytes of interest unequivocally in presence of other chemicalsubstances that may be present in the sample.

The data obtained in the ¹H NMR analyses were as follows:

Chemical shift, Sample Composition ppm I, solvent D₂O 4.79 II, chemicalshift reference D₂O-TSP 0.00 III, internal standard D₂O-DMMA 1.42 IV,GAG sample D₂O-TSP- 1.8-8.0 DMMA-GAG

The data obtained in the ¹H ¹³C-HSQC analyses were as follows:

Chemical shift Chemical shift Sample Composition ¹H, ppm ¹³C, ppm III,internal standard D₂O-DMMA 1.42 25.42 IV, sample: GAG D₂O-TSP- 1.8-8.024-112 DMMA-GAG

We found no interference between chemical shift signal for the DMMA andGAG in the ¹H NMR or ¹H ¹³C-HSQC spectra (FIGS. 2 and 3). The datademonstrate that the method is capable of discriminating, withoutinterference or undue chemical shift overlap, the chemical shift signalsof the GAG from those of other products present in the sample such asthe solvent (deuterium oxide, D₂O), the internal standard (DMMA) and thechemical shift reference (TSP-d4).

Limit of Quantification and Linearity

Under these parameters, on the one hand, the minimum quantity of analytethat may be suitably quantified precisely and accurately is determinedand, on the other hand, the capacity of the method to obtain resultsdirectly (by means of mathematical transformations) proportional to theconcentration of the analyte in the sample, within an establishedinterval.

To assess the limit of quantification and the linearity (of chemicalshift signal intensity dependence upon concentration of sample) of theintegrals (for said chemical shift signal) of the DMMA, correspondingchemical shift signals were quantified. Solution comprising enoxaparinsodium (fixed concentration) and DMMA (seven different concentrations ofDMMA) were prepared as follows:

-   -   0.2 mM of DMMA: 13.5% of the working concentration    -   0.3 Mm of DMMA: 20.3% of the working concentration    -   0.76 mM of DMMA: 50% of the working concentration    -   1.2 mM of DMMA: 80.1% of the working concentration    -   1.5 mM of DMMA: 100% of the working concentration    -   1.8 mM of DMMA: 120% of the working concentration    -   2.27 mM of DMMA: 150.2% of the working concentration.

The acceptance criteria established to fulfil this linearity criterionis that in the line obtained the correlation coefficient for bothexperiments is ≥0.99. In FIGS. 4 and 5 depicts a graph of the DMMAsignal integral values vs. DMMA concentration (mM), for the ¹H NMR and¹H ¹³C-HSQC spectra. The acceptance criterion is easily fulfilled bothfor one and the other.

The lower limit of quantification for a DMMA is a concentration of about0.20 mM or ≥20 mM. Thus, the signals of the samples studied withintensity less than the intensity of the DMMA signal corresponding tothis concentration, cannot be suitably quantified and, therefore, theycannot be taken to determine the relative proportion of the residuespresent in the molecule. This means that the GAG, or at least one of itscorresponding saccharide residues, should be present in the samplecomposition at a concentration of at least about 0.2 mM or ≥20 mM.

Accuracy

This aspect of the methods determines the proximity between the valuewhich is accepted conventionally as true or reference value and theexperimental value found. To calculate the experimental value of theconcentration of the samples in analyte solution, you correlate thesignal integral of the analyte to the signal integral of the DMMA, forexample the equation defining the linearity (described above) of signalintensity versus concentration for DMMA can be used to determine thecorresponding concentration of analyte in the sample.

The accuracy is expressed as recovery percentage in the value of a knownquantity of internal standard:

${{Recovery}\mspace{14mu}{percentage}\mspace{11mu}(R)} = {\frac{Xm}{\mu} \times 100}$Where: Xm is mean value found, and μ is the value accepted as true.

The acceptance criteria established is that the recovery values arebetween 70.0-130.0% for the concentration corresponding to theconcentration limit and 80.0-120.0% for the other levels.

The data obtained for the ¹H NMR and HSQC experiments were thefollowing:

-   a) ¹H NMR

Concentration, mM Conc. Calculated, mM Recovery, ¹H NMR 0.204 0.228111.77 0.307 0.330 107.31 0.758 0.737 97.26 1.212 1.175 96.90 1.5141.488 98.29 1.817 1.810 99.65 2.274 2.318 101.94

-   b) HSQC

Concentration, mM Conc. Calculated, mM Recovery, HSQC 0.204 0.243 119.170.307 0.337 109.81 0.758 0.727 95.96 1.212 1.140 94.03 1.514 1.488 98.291.817 1.823 100.32 2.274 2.328 102.37

Both the ¹H NMR and HSQC methods provide accuracy in compliance with theacceptance criteria for the accuracy parameters for those signalscorresponding to the sample, with intensity higher than that of thelimit of quantification.

Precision—Repeatability—Reproducibility

The intra-sample variability of the method is studied by performing aseries of analyses on the same sample in the same operating conditionsin a same laboratory and in a short period of time.

To do this, three consecutive analyses were performed for eachconcentration. The repeatability of a method is expressed as thecoefficient of variation (CV) of a series of measurements and ismathematically calculated as follows:

${{{CV}\mspace{11mu}(\%)} = {\frac{s}{X} \times 100}},$where: s is standard deviation, and X is arithmetic means of theresults.

The acceptance criterion established to fulfill these accuracy criteriais a coefficient of variation for all levels of ≤7%.

The data obtained for both experiments was the following:

Concentration, mM CV, % ¹H NMR CV, % HSQC 0.204 5.55 3.40 0.307 0.092.29 0.758 1.62 1.45 1.212 1.74 2.52 1.514 1.41 1.01 1.817 2.16 3.992.274 2.30 2.73

Both the ¹H NMR and HSQC methods provide reproducibility in compliancewith the acceptance criterion for the accuracy parameters for thosesignals corresponding to the sample, with intensity higher than that ofthe limit of quantification.

EXAMPLES

The following specific examples provided below serve to illustrate thenature of the present invention. These examples are included only forillustration purposes and are not to be deemed to limit the invention tojust said exemplary embodiments. The invention is defined by the claims,drawings, abstract and entire specification.

Example 1 ¹H NMR of Enoxaparin Sodium

Enoxaparin sodium (50 mg) are dissolved in 500 μL of a D₂O-TSP (solutionB) solution. Then 100 μL of DMMA solution (solution C) are added. Theresulting solution is introduced in a 5 mm diameter tube.

The resulting DMMA concentration in the solution is 1.5 mM. Theexperiments are performed on a Bruker AVIII-800 nuclear magneticresonance spectrometer. The main signals identified are as follows:

Signal Chemical shift, ppm H4 ΔU2S 5.992 H4 ΔU2 5.825 H1 1,6-AnA 5.616H1 ANS(-G) 5.585 H1 1,6-AnM 5.569 H1 ΔU2S 5.509 H1 ANS6S 5.405 H1 I2S5.228 H1 I 5.012 H5 I2S 4.836 H1 G 4.628 H6 ANS6S 4.344 H6′ ANS6S 4.210H3 ANS 3.670 H2 ANS3S 3.395 H2 ANS 3.293 NAc 2.047 DMMA 1.320 TSP 0.069

Once the values of the integrals of the signals both of DMMA and therest of the residues have been obtained, the normalized values areobtained of said residues dividing the value of its integrals by thevalue of the integral corresponding to the DMMA signal. Thisnormalization can be performed, because the concentration of theinternal standard is kept constant with respect to the saccharideresidue concentration for all experiments, thus avoiding theinter-experimental variability that may arise in the analysis of aseries of several product batches.

Once the normalized values of the residues have been obtained, therelative percentage of each one of them is calculated in accordance withthe following formula:

${\%\mspace{14mu}{signal}\mspace{14mu} X} = {\frac{{normalized}\mspace{14mu}{value}\mspace{14mu}{signal}\mspace{14mu} X}{\Sigma\mspace{14mu}{normalized}\mspace{14mu}{value}\mspace{14mu}{all}\mspace{14mu}{the}\mspace{14mu}{signals}} \times 100}$

To clarify the steps performed, the results obtained are shown for aseries of four samples M1, M2, M3 and M4 of enoxaparin sodium. Thefollowing integral values of each one of the chemical shift signalsselected were obtained.

Integral Signal M 1 M 2 M 3 M 4 H4 ΔU2S 2767732.83 2988384.02 3075705.312332763.55 H4 ΔU 114294.94 135658.19 143037.09 94201.86 H1 1,6-an.A404060.73 472871.53 509930.86 351978.17 H1 1,6-an.M 2535227.202700027.94 2844751.53 2160178.72 H1 ΔU2S 3541377.06 3818336.163951288.34 2999870.34 H1 ANS6S 10350026.69 10716882.48 10780945.338442504.14 H1 I2S 8922576.12 9202191.38 9486264.66 7346708.47 H5 I2S8924825.12 9540022.53 10041127.64 7751650.03 H2 ANS 16351247.2216802874.61 17051664.14 13189968.22 NAc 8054931.36 7973188.48 8124533.566388424.34 DMMA 1464255.27 1415020.17 1485242.95 1279612.94where M corresponds to sample.

To obtain the normalized values of the integrals of the chemical shiftsignals, their respective integrals are divided by the integral of theDMMA (present at a known or determined concentration):

Normalized integral Signal M 1 M 2 M 3 M 4 H4 ΔU2S 1.890 2.112 2.0711.823 H4 ΔU 0.078 0.096 0.096 0.074 H1 1,6-an.A 0.276 0.334 0.343 0.275H1 1,6-an.M 1.731 1.908 1.915 1.688 H1 ΔU2S 2.419 2.698 2.660 2.344 H1ANS6S 7.068 7.574 7.259 6.598 H1 I2S 6.094 6.503 6.387 5.741 H5 I2S6.095 6.742 6.761 6.058 H2 ANS 11.167 11.875 11.481 10.308 NAc 5.5015.635 5.470 4.992 DMMA 1.000 1.000 1.000 1.000

From these normalized values, the relative proportion of each one of thechemical shift signals is calculated with respect to the sum of all thenormalized chemical shift signals.

Relative proportion, % Signal M 1 M 2 M 3 M 4 H4 ΔU2S 4.47 4.64 4.664.57 H4 ΔU 0.18 0.21 0.22 0.18 H1 1,6-an.A 0.65 0.73 0.77 0.69 H11,6-an.M 4.09 4.20 4.31 4.23 H1 ΔU2S 5.72 5.93 5.99 5.88 H1 ANS6S 16.7016.65 16.33 16.54 H1 I2S 14.40 14.30 14.37 14.39 H5 I2S 14.40 14.8315.21 15.18 H2 ANS 26.39 26.11 25.83 25.83 NAc 13.00 12.39 12.31 12.51

The quantification of the characteristic and well-differentiated signalsof enoxaparin sodium (generally those corresponding to the anomeric ortarget protons, H1) are shown in the following table with the observedrelative proportion values:

Signal Chemical shift, ppm Relative proportion, % H4 ΔU2S 5.99 4.3-4.7H4 ΔU 5.82 0.2 H1 1,6-an.A 5.62 0.7-0.9 H1 1,6-an.M 5.57 4.1-4.4 H1 ΔU2S5.51 5.7-6.0 H1 ANS6S 5.40 16.1-16.7 H1 I2S 5.23 13.1-14.4 H5 I2S 4.8414.3-16.3 H2 ANS 3.29 24.3-26.6 NAc 2.05 12.0-15.3

The set of the signals corresponding to the peaks found in a determinedNMR spectrum, whether one-dimensional ¹H-NMR and/or two-dimensional¹H-¹³C HSQC, or the absence thereof, in the relative proportions of itsnormalized integrals indicated by the parameter “relative proportion(%)” is what in the present specification is called “signal pattern” orsimply “pattern”.

Example 2

The same solution used in Example 1 is used to perform the study by¹H-¹³C HSQC. The main signals identified are as follows:

Signal ppm δ ¹³C, ppm δ ¹H, ppm C4-H4 ΔU 110.71 5.82 C4-H4 ΔU2S 108.975.99 C1-H1 G // Gal 106.62 4.66 C1-H1 Xyl 105.79 4.45 C1-H1 G(-ANAc)105.13 4.50 C1-H1 I(-A6S) 104.94 5.01 C1-H1 G(-ANS) 104.77 4.60 C1-H1I(-A6OH) 104.67 4.94 C1-H1 Gal 104.30 4.54 C1-H1 1,6-an.A 104.22 5.61C1-H1 G(-ANS3S) 103.91 4.61 C1-H1 1,6-an.M 103.91 5.57 C1-H1 ΔU 103.885.16 C1-H1 G2S 102.99 4.75 C1-H1_I2S 102.09 5.22 C1-H1 I2S(-1,6-an.M)101.59 5.36 C1-H1 ANS(-G) 100.50 5.58 C1-H1 ANAc 100.23 5.31 C1-H1 ΔU2S100.18 5.51 C1-H1 ANS(-I2S) 99.78 5.40 C1-H1 ANS6S 99.43 5.43 C1-H1ANS,3S 99.06 5.51 C1-H1 ANS βred 98.73 4.71 C1-H1 ANS(-I) 98.42 5.34C1-H1 M αred 95.74 5.39 C1-H1 I2S αred 95.70 5.42 C1-H1 I2S βred 94.764.97 C1-H1 ANS αred// 93.97 5.45 ANS6S red C3-H3 Gal 85.45 3.78 C3-H3Gal 84.85 3.83 C4-H4 ANS6S(-G)// 80.94 3.84 ANS6S red C2-H2 I2S 78.534.34 C3-H3 Xyl 77.82 3.72 C2-H2 ΔU2S 77.42 4.62 C2-H2 G(-AN6S) 75.703.40 C3-H3 ANS6S (-G) 72.47 3.66 C3-H3 ANS6S red 72.29 3.77 C5-H5 I2S72.01 4.83 C3-H3 I2S 71.87 4.21 C5-H5 ANS6S(-G) 71.72 4.09 C5-H5 MNS6Sred 70.98 4.15 C5-H5 ANS6S red 70.64 4.12 C6-H6 1,6-an.A// 67.53 3.771,6-an.M C5-H5 Xyl 65.89 4.12 C5-H5 Xyl 65.86 3.40 C3-H3 ΔU2S 65.75 4.32C6-H6 Gal 63.90 3.74 C2-H2 ANS6S red// 60.82 3.28 ANS(-I2S) C2-H2ANS6S(-G) 60.52 3.29 C2-H2 MNS6S red 60.38 3.60 C2-H2 1,6-an.A 58.503.21 C2-H2 ANAc 56.68 3.92 C2-H2 1,6-an.M 55.09 3.47 DMMA 26.73 1.32 NAc24.87 2.05

These signals are then correlated with monosaccharide components of themolecule, so that their quantification provides a chemical shift signalpattern representative of the monosaccharide content of the GAG.

The integral for each one of these ¹³C chemical shift signals wasnormalized from the value set for the integral of the reference ¹³Cchemical shift signal of DMMA, using the same process (calculations)described for the ¹H NMR experiments. The quantification of thecharacteristic signals of enoxaparin sodium is shown in the followingtable:

Signal Relative proportion, % C1-H1 ANS-I2S 25.6-26.9 C1-H1 ANS-I2.6-3.0 C1-H1 ANS-G 5.1-5.5 C1-H1 ANS.3S 1.5-1.7 C1-H1 ANAc 2.7-3.5C1-H1 ANAc-αred <LC C1-H1 ANS-red 3.8-4.9 C1-H1 1,6-an.A 1.2-1.5 C1-H11,6-an.M 1.6-1.9 C1-H1 MNS-αred 1.0-1.3 C1-H1 I2S 24.5-27.5 C1-H1 I-A6S2.4-2.7 C1-H1 I-A6OH 0.3-0.4 C1-H1 G-ANS.3S 1.4-1.6 C1-H1 G-ANS 4.2-4.4C1-H1 G-ANAc 1.9-2.6 C1-H1 G2S 1.1-1.6 C1-H1 ΔU2S 11.5-12.4 C1-H1 ΔU0.3-0.5 C1-H1 I2S-red 1.0-1.4 C5-H5 Gal-A <LC-0.5  Epox <LC-0.4 

These experiments demonstrate that, using the experimental conditionsdescribed above, it is possible to obtain an analysis method by nuclearmagnetic resonance (¹H-NMR and ¹H-¹³C HSQC) of glycosaminoglycans ingeneral, and of heparins and low molecular weight heparins and theirderivatives in particular, which allows their quantitative analysis.

Example 3 Study by ¹H NMR of Bemiparin Sodium

The main ¹H chemical shift signals identified were as follows:

Chemical Signal shift, ppm H4 ΔU2S 5.992 H4 ΔU 5.825 H1 1,6-AnA 5.616 H1ANS(-G) 5.585 H1 1,6-AnM 5.569 H1 ΔU2S 5.509 H1 ANS6S 5.405 H1 I2S 5.228H1 I 5.012 H5 I2S 4.836 H1 G 4.628 H6 ANS6S 4.344 H6′ ANS6S 4.210 H3 ANS3.670 H2 ANS3S 3.395 H2 ANS 3.293 NAc 2.047 DMMA 1.320 TSP 0.069

The quantification of the characteristic and well-differentiated signalsof bemiparin sodium (generally those corresponding to the anomeric ortarget protons, H1) are shown in the following table, with the values ofrelative proportions observed for a series of six samples.

Signal Chemical shift, ppm Relative proportion, % H4 ΔU2S 5.99 3.7-5.7H4 ΔU 5.82 0.2-2.5 H1 1,6-an.A 5.62 0.5-2.5 H1 1,6-an.M 5.57 2.5-6.0 H1ΔU2S 5.51  7.0-10.7 H1 ANS6S 5.40 19.0-21.3 H1 I2S 5.23 13.8-18.5 H2 ANS3.29 18.7-26.3 NAc 2.05  9.4-14.4

Example 4

The same solution used in example 3, is used to perform the study by¹H-¹³C HSQC. The main ¹H and ¹³C chemical shift signals identified wereas follows:

Signal ppm δ ¹³C, ppm δ ¹H, ppm C4-H4 ΔU 110.71 5.82 C4-H4 ΔU2S 108.975.99 C1-H1 G // Gal 106.62 4.66 C1-H1 Xyl 105.79 4.45 C1-H1 G(-ANAc)105.13 4.50 C1-H1 I(-A6S) 104.94 5.01 C1-H1 G(-ANS) 104.77 4.60 C1-H1I(-A6OH) 104.67 4.94 C1-H1 Gal 104.30 4.54 C1-H1 1.6-an.A 104.22 5.61C1-H1 G(-ANS3S) 103.91 4.61 C1-H1 1.6-an.M 103.91 5.57 C1-H1 ΔU 103.885.16 C1-H1 G2S 102.99 4.75 C1-H1_I2S 102.09 5.22 C1-H1 I2S(-1,6-an.M)101.59 5.36 C1-H1 ANS(-G) 100.50 5.58 C1-H1 ANAc 100.23 5.31 C1-H1 ΔU2S100.18 5.51 C1-H1 ANS(-I2S) 99.78 5.40 C1-H1 ANS6S 99.43 5.43 C1-H1ANS,3S 99.06 5.51 C1-H1 ANS βred 98.73 4.71 C1-H1 ANS(-I) 98.42 5.34C1-H1 M αred 95.74 5.39 C1-H1 I2S αred 95.70 5.42 C1-H1 I2S βred 94.764.97 C1-H1 ANS αred// 93.97 5.45 ANS6S red C3-H3 Gal 85.45 3.78 C3-H3Gal 84.85 3.83 C4-H4 ANS6S(-G)// 80.94 3.84 ANS6S red C2-H2 I2S 78.534.34 C3-H3 Xyl 77.82 3.72 C2-H2 ΔU2S 77.42 4.62 C2-H2 G(-AN6S) 75.703.40 C3-H3 ANS6S (-G) 72.47 3.66 C3-H3 ANS6S red 72.29 3.77 C5-H5 I2S72.01 4.83 C3-H3 I2S 71.87 4.21 C5-H5 ANS6S(-G) 71.72 4.09 C5-H5 MNS6Sred 70.98 4.15 C5-H5 ANS6S red 70.64 4.12 C6-H6 1,6-an.A// 67.53 3.771,6-an.M C5-H5 Xyl 65.89 4.12 C5-H5 Xyl 65.86 3.40 C3-H3 ΔU2S 65.75 4.32C6-H6 Gal 63.90 3.74 C2-H2 ANS6S red// 60.82 3.28 ANS(-I2S) C2-H2ANS6S(-G) 60.52 3.29 C2-H2 MNS6S red 60.38 3.60 C2-H2 1.6-an.A 58.503.21 C2-H2 ANAc 56.68 3.92 C2-H2 1.6-an.M 55.09 3.47 DMMA 26.73 1.32 NAc24.87 2.05

The signals were correlated with particular saccharides (based uponcomparison of said signals to those of reference monosaccharides), andafter normalization of the respective integrals of said signals withrespect to the integral of the DMMA reference, and after determining therelative percentage of the individual signals, a quantitative chemicalshift pattern for the GAG was obtained.

Specifically, the integral of each one of the above signals wasnormalized with respect to the integral of DMMA, using the sameprocedure explained for the experiments ¹H MMR. Accordingly, thequantitative relative content (proportion) of each saccharide inbemiparin sodium is detailed in the following table.

Signal Relative proportion, % C1-H1 ANS-I2S 26.5-30.6 C1-H1 ANS-I1.7-5.3 C1-H1 ANS-G 2.1-3.8 C1-H1 ANS.3S 0.6-2.5 C1-H1 ANAc 1.7-3.0C1-H1 ANAc-αred <LC C1-H1 ANS-red 2.6-5.4 C1-H1 1,6-an.A <1.1 C1-H11,6-an.M <1.0 C1-H1 MNS-αred 0.9-2.3 C1-H1 I2S 30.4-34.9 C1-H1 I-A6S1.4-2.6 C1-H1 I-A6OH <0.2 C1-H1 G-ANS,3S <2.5 C1-H1 G-ANS 1.9-3.6 C1-H1G-ANAc 0.4-1.4 C1-H1 G2S <0.5 C1-H1 ΔU2S 10.9-14.9 C1-H1 ΔU 0.6-1.6C1-H1 I2S-red <0.5 C5-H5 Gal-A <0.3

Example 5 Study by ¹H NMR of Dalteparin Sodium

The main ¹H chemical shift signals identified were as follows.

Chemical Signal shift, ppm H1 ANS(-G) 5.585 H1 ANS6S 5.405 H1 I2S 5.228H1 I2S-(AM.ol) 5.178 H1 I 5.012 H5 I2S 4.836 H1 G 4.628 H6 ANS6S 4.344H6′ ANS6S 4.210 H3 ANS 3.670 H2 ANS3S 3.395 H2 ANS 3.293 NAc 2.047 DMMA1.320 TSP 0.069

After normalization, correlation and relative content determination, thequantitative relative content (proportion) of each saccharide indalteparin sodium was obtained and is detailed in the following table.The quantitation (based upon a series of six samples) is based upon thethe characteristic and well-differentiated signals of dalteparin sodium(generally those corresponding to the anomeric or target protons, H1).

Signal Chemical shift, ppm Relative proportion, % H1 ANS6S 5.4025.5-25.8 H1 I2S 5.23 19.2-20.8 H1 I2S-(AM.ol) 5.18 9.5-9.8 H2 ANS 3.2928.0-30.0 NAc 2.05 15.4-20.0

Example 6

The same solution used in example 5 was used to perform the study by¹H-¹³C HSQC. The main ¹H and ¹³C chemical shift signals identified wereas follows:

Signal δ ¹³C, ppm δ ¹H, ppm C1-H1 G // Gal 106.62 4.66 C1-H1 Xyl 105.794.45 C1-H1 G(-ANAc) 105.13 4.50 C1-H1 I(-A6S) 104.94 5.01 C1-H1 G(-ANS)104.77 4.60 C1-H1 I(-A6OH) 104.67 4.94 C1-H1 G(-ANS3S) 103.91 4.61 C1-H1G2S 102.99 4.75 C1-H1 I2S 102.09 5.22 C1-H1 ANS(-G) 100.50 5.58 C1-H1ANAc 100.23 5.31 C1-H1 ANS(-I2S) 99.78 5.40 C1-H1 ANS6S 99.43 5.43 C1-H1ANS,3S 99.06 5.51 C1-H1 ANS(-I) 98.42 5.34 C3-H3 Gal 85.45 3.78 C3-H3Gal 84.85 3.83 C4-H4 ANS6S(-G) 80.94 3.84 C2-H2 I2S 78.53 4.34 C3-H3 Xyl77.82 3.72 C2-H2 G(-AN6S) 75.70 3.40 C3-H3 ANS6S (-G) 72.47 3.66 C5-H5I2S 72.01 4.83 C3-H3 I2S 71.87 4.21 C5-H5 ANS6S(-G) 71.72 4.09 C5-H5 Xyl65.89 4.12 C5-H5 Xyl 65.86 3.40 C6-H6 Gal 63.90 3.74 AM.ol-6S 63.8/63.73.70/3.74 C2-H2 ANS(-I2S) 60.82 3.28 C2-H2 ANS6S(-G) 60.52 3.29 C2-H2ANAc 56.68 3.92 DMMA 26.73 1.32 NAc 24.87 2.05

These signals can be associated with the monosaccharide components ofthe molecule, so that their quantification allows for the determinationof their monosaccharide composition.

The integrals of each one of these signals were normalized starting fromthe value established for the integral of DMMA, using the same procedureexplained for the experiments ¹H NMR. The quantification of the signalscharacteristic of dalteparin sodium are shown in the following table.

Signal Relative proportion, % C1-H1 ANS-I2S 22.2-23.3 C1-H1 ANS-I3.0-3.2 C1-H1 ANS-G 2.3-2.6 C1-H1 ANS,3S 2.1-2.9 C1-H1 ANAc 2.4-3.1C1-H1 I2S 24.5-27.5 C1-H1 I-A6S 3.6-4.0 C1-H1 G-ANS,3S 1.8-2.3 C1-H1G-ANS 2.5-3.5 C1-H1. C6-H6 AM.ol-6S 20.8-21.7

Example 7 Study by ¹H NMR of Tinzaparin Sodium

The main ¹H chemical shift signals identified were as follows:

Chemical Signal shift, ppm H4 ΔU2S 5.992 H4 ΔU 5.825 H1 ANS(-G) 5.585 H1ΔU2S 5.509 H1 ANS6S 5.405 H1 I2S 5.228 H1 I 5.012 H5 I2S 4.836 H1 G4.628 H6 ANS6S 4.344 H6′ ANS6S 4.210 H3 ANS 3.670 H2 ANS3S 3.395 H2 ANS3.293 NAc 2.047 DMMA 1.320 TSP 0.069

The quantification of the characteristic and well-differentiated signalsof tinzaparin sodium (generally those corresponding to the anomeric ortarget protons, H1) are shown in the following table, with the values ofrelative proportions observed (based upon a series of six samples).

Signal Chemical shift, ppm Relative proportion, % H4 ΔU2S 5.99 2.7 H1ΔU2S 5.51 5.3 H1 ANS6S 5.40 23.6 H1 I2S 5.23 21.0 H2 ANS 3.29 30.0 NAc2.05 16.1

Example 8

The same solution used in example 7 was used to perform the study by¹H-¹³C HSQC. The main signals identified were as follows:

Signal ppm δ ¹³C, ppm δ ¹H, ppm C4-H4 ΔU 110.71 5.82 C4-H4 ΔU2S 108.975.99 C1-H1 G // Gal 106.62 4.66 C1-H1 Xyl 105.79 4.45 C1-H1 G(-ANAc)105.13 4.50 C1-H1 I(-A6S) 104.94 5.01 C1-H1 G(-ANS) 104.77 4.60 C1-H1I(-A6OH) 104.67 4.94 C1-H1 Gal 104.30 4.54 C1-H1 G(-ANS3S) 103.91 4.61C1-H1 ΔU 103.88 5.16 C1-H1 G2S 102.99 4.75 C1-H1 I2S 102.09 5.22 C1-H1ANS(-G) 100.50 5.58 C1-H1 ANAc 100.23 5.31 C1-H1 ΔU2S 100.18 5.51 C1-H1ANS(-I2S) 99.78 5.40 C1-H1 ANS6S 99.43 5.43 C1-H1 ANS,3S 99.06 5.51C1-H1 ANS βred 98.73 4.71 C1-H1 ANS(-I) 98.42 5.34 C1-H1 I2S αred 95.705.42 C1-H1 I2S βred 94.76 4.97 C1-H1 ANS αred// 93.97 5.45 ANS6S redC3-H3 Gal 85.45 3.78 C3-H3 Gal 84.85 3.83 C4-H4 ANS6S(-G)// 80.94 3.84ANS6S red C2-H2 I2S 78.53 4.34 C3-H3 Xyl 77.82 3.72 C2-H2 ΔU2S 77.424.62 C2-H2 G(-AN6S) 75.70 3.40 C3-H3 ANS6S (-G) 72.47 3.66 C3-H3 ANS6Sred 72.29 3.77 C5-H5 I2S 72.01 4.83 C3-H3 I2S 71.87 4.21 C5-H5 ANS6S(-G)71.72 4.09 C5-H5 ANS6S red 70.64 4.12 C5-H5 Xyl 65.89 4.12 C5-H5 Xyl65.86 3.40 C3-H3 ΔU2S 65.75 4.32 C6-H6 Gal 63.90 3.74 C2-H2 ANS6S red//60.82 3.28 ANS(-I2S) C2-H2 ANS6S(-G) 60.52 3.29 C2-H2 MNS6S red 60.383.60 C2-H2 ANAc 56.68 3.92 DMMA 26.73 1.32 NAc 24.87 2.05

These signals can be associated with the monosaccharide components ofthe molecule, so that their quantification allows the determination oftheir monosaccharide composition.

The integrals of each one of these signals were normalized starting fromthe value established for the integral of DMMA, using the same procedureexplained for the experiments ¹H RMN. The quantification of the signalscharacteristic of tinzaparin sodium are shown in the following table:

Signal Relative proportion, % C1-H1 ANS-I2S 27.2 C1-H1 ANS-I 3.2 C1-H1ANS-G 3.4 C1-H1 ANS.3S 1.2 C1-H1 ANAc 3.7 C1-H1 ANAc-αred <LC C1-H1ANS-red 6.5 C1-H1 I2S 35.1 C1-H1 I-A6S 3.1 C1-H1 I-A6OH 0.8 C1-H1G-ANS,3S 1.5 C1-H1 G-ANS 3.4 C1-H1 G-ANAc 2.2 C1-H1 ΔU2S 8.6 C1-H1I2S-red <0.1

These experiments show that, using the above-described experimentalconditions, it is possible to obtain a method of analysis by nuclearmagnetic resonance (¹H-RMN y ¹H-¹³C HSQC) of glycosaminoglycans ingeneral and of heparins and low molecular weight heparins and theirderivatives in particular, which allows their quantitative analysis.

Additional Disclosure

The invention includes at least the following embodiments.

A method for the analysis of a composition that contains monosaccharideresidues present in heparin chains by means of ¹H-NMR one-dimensionalnuclear magnetic resonance and/or ¹H-¹³C HSQC two-dimensional nuclearmagnetic resonance comprising the steps of:

-   providing a composition including at least one monosaccharide    residue present in heparin chains and obtaining its spectrum of    ¹H-NMR one-dimensional nuclear magnetic resonance and/or ¹H-¹³C HSQC    two-dimensional nuclear magnetic resonance using dimethylmalonic    acid (DMMA) as internal reference, and-   identifying in the NMR spectrum the presence or the absence of at    least one signal of at least one residue selected from the group    consisting of: 4,5-unsaturated 2-O sulfo uronic acid (ΔU2S),    4,5-unsaturated uronic acid (ΔU), 2-N-sulfo-1,6-anhydroglucosamine    (1,6-an.A), 2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M),    2-N-sulfo-6-O-sulfoglucosamine (ANS6S), 2,5-anhydro mannitol,    N-sulfoglucosamine, glucuronic acid, N-sulfo-6-O-sulfoglucosamine,    2-O-sulfoiduronic acid, iduronic acid, N-sulfo-3-O-sulfoglucosamine,    N-sulfo-3.6-di-O-sulfoglucosamine, galacturonic acid, Xylose,    N-acetylglucosamine and N-acetyl-6-O-sulfoglucosamine,-   characterized in that the presence of said NMR signals in a    determined relative proportion of its normalized integrals with    respect to DMMA, or the absence thereof, forms a pattern which    allows identifying the heparin which the monosaccharide residue    comes from comparing the pattern obtained in the analysis with a    standard pattern previously obtained for different heparins in the    same conditions.

The method according to any of the embodiments herein, wherein thefollowing pattern is identified in the ¹H NMR spectrum:

Signal Chemical shift, ppm Relative proportion, % H4 ΔU2S 5.99 4.3-4.7H4 ΔU 5.82 0.2 Hl 1,6-an.A 5.62 0.7-0.9 Hl 1,6-an.M 5.57 4.1-4.4 Hl ΔU2S5.51 5.7-6.0 Hl ANS6S 5.40 16.1-16.7 Hl I2S 5.23 13.1-14.4 H5 I2S 4.8414.3-16.3 H2 ANS 3.29 24.3-26.6 NAc 2.05 12.0-15.3thereby determining the content of the monosaccharide residues inenoxaparin sodium.

The method according to any of the embodiments herein, wherein thefollowing pattern is identified in the ¹H-¹³C HSQC NMR spectrum:

Signal Relative proportion, % C1-H1 ANS-I2S 25.6-26.9 C1-H1 ANS-I2.6-3.0 C1-H1 ANS-G 5.1-5.5 C1-H1 ANS.3S 1.5-1.7 C1-H1 ANAc 2.7-3.5C1-H1 ANAc-αred <LC C1-H1 ANS-red 3.8-4.9 C1-H1 1,6-an.A 1.2-1.5 C1-H11,6-an.M 1.6-1.9 C1-H1 MNS-αred 1.0-1.3 C1-H1 I2S 24.5-27.5 C1-H1 I-A6S2.4-2.7 C1-H1 I-A6OH 0.3-0.4 C1-H1 G-ANS.3S 1.4-1.6 C1-H1 G-ANS 4.2-4.4C1-H1 G-ANAc 1.9-2.6 C1-H1 G2S 1.1-1.6 C1-H1 ΔU2S 11.5-12.4 C1-H1 ΔU0.3-0.5 C1-H1 I2S-red 1.0-1.4 C5-H5 Gal-A <LC-0.5  Epox <LC-0.4 where “LC” is “limit of quantification”,thereby determining the content of the monosaccharide residues inenoxaparin sodium.

The method according to any of the embodiments herein, wherein thefollowing pattern is identified in the ¹H NMR spectrum:

Signal Chemical shift, ppm Relative proportion, % H4 ΔU2S 5.99 3.7-5.7H4 ΔU 5.82 0.2-2.5 H1 1,6-an.A 5.62 0.5-2.5 H1 1,6-an.M 5.57 2.5-6.0 H1ΔU2S 5.51  7.0-10.7 H1 ANS6S 5.40 19.0-21.3 H1 I2S 5.23 13.8-18.5 H2 ANS3.29 18.7-26.3 NAc 2.05 9.4-14 thereby determining the content of the monosaccharide residues inbemiparin sodium.

The method according to any of the embodiments herein, wherein thefollowing pattern is identified in the ¹H-¹³C HSQC NMR spectrum:

Signal Relative proportion, % C1-H1 ANS-I2S 26.5-30.6 C1-H1 ANS-I1.7-5.3 C1-H1 ANS-G 2.1-3.8 C1-H1 ANS.3S 0.6-2.5 C1-H1 ANAc 1.7-3.0C1-H1 ANAc-αred <LC C1-H1 ANS-red 2.6-5.4 C1-H1 1,6-an.A <1.1 C1-H11,6-an.M <1.0 C1-H1 MNS-αred 0.9-2.3 C1-H1 I2S 30.4-34.9 C1-H1 I-A6S1.4-2.6 C1-H1 I-A6OH <0.2 C1-H1 G-ANS,3S <2.5 C1-H1 G-ANS 1.9-3.6 C1-H1G-ANAc 0.4-1.4 C1-H1 G2S <0.5 C1-H1 ΔU2S 10.9-14.9 C1-H1 ΔU 0.6-1.6C1-H1 I2S-red <0.5 C5-H5 Gal-A <0.3thereby determining the content of the monosaccharide residues inbemiparin sodium.

The method according to any of the embodiments herein, wherein thefollowing pattern is identified in the ¹H NMR spectrum:

Signal Chemical shift, ppm Relative proportion, % H1 ANS6S 5.4025.5-25.8 H1 I2S 5.23 19.2-20.8 H1 I2S-(AM.ol) 5.18 9.5-9.8 H2 ANS 3.2928.0-30.0 NAc 2.05 15.4-20.0thereby determining the content of the monosaccharide residues indalteparin sodium.

The method according to any of the embodiments herein, wherein thefollowing pattern is identified in the ¹H-¹³C HSQC NMR spectrum:

Signal Relative proportion, % C1-H1 ANS-I2S 22.2-23.3 C1-H1 ANS-I3.0-3.2 C1-H1 ANS-G 2.3-2.6 C1-H1 ANS,3S 2.1-2.9 C1-H1 ANAc 2.4-3.1C1-H1 I2S 24.5-27.5 C1-H1 I-A6S 3.6-4.0 C1-H1 G-ANS.3S 1.8-2.3 C1-H1G-ANS 2.5-3.5 C1-H1. C6-H6 AM.ol-6S 20.8-21.7thereby determining the content of the monosaccharide residues indalteparin sodium.

The method according to any of the embodiments herein, wherein thefollowing pattern is identified in the ¹H NMR spectrum:

Signal Chemical shift, ppm Relative proportion, % H4 ΔU2S 5.99 2.7 H1ΔU2S 5.51 5.3 H1 ANS6S 5.40 23.6 H1 I2S 5.23 21.0 H2 ANS 3.29 30.0 NAc2.05 16.1thereby determining the content of the monosaccharide residues intinzaparin sodium.

The method according to any of the embodiments herein, wherein thefollowing pattern is identified in the ¹H-¹³C HSQC NMR spectrum:

Signal Relative proportion, % C1-H1 ANS-I2S 27.2 C1-H1 ANS-I 3.2 C1-H1ANS-G 3.4 C1-H1 ANS,3S 1.2 C1-H1 ANAc 3.7 C1-H1 ANAc-αred <LC C1-H1ANS-red 6.5 C1-H1 I2S 35.1 C1-H1 I-A6S 3.1 C1-H1 I-A6OH 0.8 C1-H1G-ANS,3S 1.5 C1-H1 G-ANS 3.4 C1-H1 G-ANAc 2.2 C1-H1 ΔU2S 8.6 C1-H1I2S-red <0.1thereby determining the content of the monosaccharide residues intinzaparin sodium.

The method according to any of the embodiments herein, wherein thesignals corresponding to the N-acetyl groups appear in the regionbetween 1.8 to 2.1 ppm in of ¹H-NMR spectroscopy.

The method according to any of the embodiments herein, wherein thesignals corresponding to the saccharide ring of said residues appear inthe region between 2.8 to 6.0 ppm in ¹H-NMR spectroscopy.

The method according to any of the embodiments herein, wherein thesignals corresponding to the anomeric or target H1 protons, and that ofthe H4 protons of the non-reducing ends of one of said residues, appearin the region between 4.6 to 6.0 ppm in ¹H-NMR spectroscopy.

The method according to any of the embodiments herein, wherein, in ¹HNMR spectroscopy, the 4,5-unsaturated 2-O-sulfo-uronic acid (ΔU2S)signals appear at 5.99 and 5.51 ppm, for the H4 and anomeric protonsrespectively, the 4,5-unsaturated uronic acid (ΔU) signal appears at5.82 ppm for the H4 proton, the 2-N-sulfo-1,6-anhydroglucosamine(1,6-an.A) signal appears at 5.62 ppm for the anomeric proton, the2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M) signal appears at 5.57 ppmfor the anomeric proton, and the 2-N-sulfo-6-O-sulfoglucosamine signalsappear at 5.41 and 4.21-4.34 ppm for the H1 and H6 and H6′ protonsrespectively.

The method according to any of the embodiments herein, wherein, in¹H-¹³C HSQC NMR spectroscopy, the 4,5-unsaturated 2-O sulfo uronic acid(ΔU2S) signals appear at 6.0-109.0 ppm (H4-C4), 5.5-100.2 ppm (H1-C1),4.6-77.4 ppm (H2-C2) or 4.3-66.8 ppm (H3-C3), the 4,5-unsaturated uronicacid (ΔU) signals appear at 5.8-110.7 ppm (H4-C4) or 5.2-103.9 ppm(H1-C1), the 2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A) signals appearat 5.6-104.2 ppm (H1-C1), 3.2-58.5 ppm (H2-C2) or 3.8-67.5 ppm (H6-C6),the 2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M) signals appear at5.6-103.9 ppm (H1-C1), 3.5-55.1 (H2-C2) or 3.8-67.5 ppm (H6-C6), and the2-N-sulfo-6-O-sulfoglucosamine signals appear at 5.4-99.4 ppm (H1-C1),3.8-80.9 ppm (H4-C4), 3.7 to 3.8-42.3 to 72.5 ppm (H3-C3), 4.1-70.6 to71.7 ppm (H5-C5) or 3.3-60.5-60.8 ppm (H2-C2).

The method according to any of the embodiments herein, wherein thepattern obtained in the analysis is such that it determines that themonosaccharide residues come from an unfractionated heparin.

The method according to any of the embodiments herein, wherein thepattern obtained in the analysis is such that it determines that themonosaccharide residues come from a Low Molecular Weight Heparin (LMWH).

The method according to any of the embodiments herein, wherein thepattern obtained in the analysis is such that it determines that themonosaccharide residues come from an Ultra Low Molecular Weight Heparin(ULMWH).

The invention according to any of the embodiments herein, wherein theglycosaminoglycan is selected from the group consisting of enoxaparin,bemiparin, dalteparin, tinzaparin, a salt of any of the preceding, aderivative of any of the preceding, or a combination thereof.

In view of the above description and the examples below, one of ordinaryskill in the art will be able to practice the invention as claimedwithout undue experimentation. The foregoing will be better understoodwith reference to the following examples that detail certain proceduresfor the preparation and/or practice of embodiments of the presentinvention. All references made to these examples are for the purposes ofillustration. The following examples should not be consideredexhaustive, but merely illustrative of only a few of the manyembodiments contemplated by the present invention.

As used herein, the term “about” or “approximately” are taken tomean±10%, ±5%, ±2.5% or ±1% of a specified valued. As used herein, theterm “substantially” is taken to mean “to a large degree” or “at least amajority of” or “more than 50% of”. Moreover, all ranges specifiedherein are inclusive of the range limits and all integer and fractionalvalues therein especially as defined by the definition of the term“about”.

As used herein a “derivative” is: a) a chemical substance that isrelated structurally to a first chemical substance and theoreticallyderivable from it; b) a compound that is formed from a similar firstcompound or a compound that can be imagined to arise from another firstcompound, if one atom of the first compound is replaced with anotheratom or group of atoms; c) a compound derived or obtained from a parentcompound and containing essential elements of the parent compound; or d)a chemical compound that may be produced from first compound of similarstructure in one or more steps. For example, a derivative may include adeuterated form, oxidized form, dehydrated, unsaturated, polymerconjugated or glycosilated form thereof or may include an ester, amide,lactone, homolog, ether, thioether, cyano, amino, alkylamino,sulfhydryl, heterocyclic, heterocyclic ring-fused, polymerized,pegylated, benzylidenyl, triazolyl, piperazinyl or deuterated formthereof.

The invention claimed is:
 1. A composition comprising: at least oneglycosaminoglycan comprising at least one saccharide comprising anomericor target hydrogen exhibiting respective anomeric or target ¹H chemicalshift signal in the range of about 3.2 ppm to about 6 ppm when analyzedby NMR; at least one reference compound comprising at least onereference hydrogen atom having a NMR signal t1 longitudinal relaxationtime of about 1 s or less, wherein the at least one reference hydrogenexhibits a reference ¹H NMR chemical shift signal separated from saidanomeric or target ¹H chemical shift signals, and said reference ¹Hchemical shift signal exhibits a concentration dependent signalintensity.
 2. The composition of claim 1, wherein said reference ¹H NMRchemical shift signal is in the range of about 1.2 to about 1.6 ppm. 3.The composition of claim 1, wherein said glycosaminoglycan is anoligosaccharide or polysaccharide.
 4. The composition of claim 1,wherein the at least one saccharide is selected from the groupconsisting of 4,5-unsaturated 2-O sulfo-uronic acid (ΔU2S),4,5-unsaturated uronic acid (ΔU), 2-N-sulfo-1,6-anhydroglucosamine(1,6-an.A), 2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M),2-N-sulfo-6-O-sulfoglucosamine (ANS6S), 2,5-anhydro mannitol,N-sulfoglucosamine, glucuronic acid, N-sulfo-6-O-sulfoglucosamine,2-O-sulfoiduronic acid, iduronic acid, N-sulfo-3-O-sulfoglucosamine,N-sulfo-3,6-di-O-sulfoglucosamine, galacturonic acid, Xylose,N-acetylglucosamine and N-acetyl-6-O-sulfoglucosamine.
 5. Thecomposition of claim 1, wherein said at least one reference compound ispresent at a concentration in the range of about 0.2 mM to about 2.5 mM.6. The composition of claim 1, wherein said reference ¹H-NMR chemicalshift signal is outside the range of 3.2 to 6 ppm.
 7. The composition ofclaim 1, wherein said reference ¹H-NMR chemical shift signal is asinglet within the range of about 1.2 ppm to about 1.6 ppm inclusive ofthe range limits.
 8. The composition of claim 1, wherein said reference¹H-NMR chemical shift is relative to the singlet chemical shift of3-(trimethylsilyl)-priopionic-D4 acid assigned as 0 ppm.
 9. Thecomposition of claim 1, wherein said at least one reference compound isdimethylmalonic acid.
 10. The composition of claim 1, wherein said atleast one reference compound is present at a known or predeterminedconcentration or amount.
 11. The composition of claim 1, wherein said atleast one glycosaminoglycan is present at a known or predeterminedconcentration or amount or at a concentration in the range of about 0.02to about 0.2 mg/μL.
 12. The composition of claim 1, wherein said atleast one glycosaminoglycan is selected from the group consisting ofheparin, heparan, enoxaparin, bemiparin, dalteparin, tinzaparin, a saltof any of the preceding, a derivative of any of the preceding, asulfated or non-sulfated form of any of the preceding, an ultra-lowmolecular weight form of any of the preceding, a low molecular weightform of any of the preceding, a high molecular weight form of any of thepreceding, an unfractionated form of any of the preceding, afractionated form of any of the preceding, and a combination of anythereof.
 13. The composition of claim 1 further comprising a chemicalshift reference standard defining 0 ppm.
 14. The composition of claim 13further comprising at least one deuterated solvent for said at least oneglycosaminoglycan, said at least one reference compound, and saidchemical shift reference standard.
 15. The composition of claim 14,wherein said deuterated solvent is selected from the group consisting ofD₂O, any solvent that will solubilize the at least one GAG and the atleast one reference compound, and a combination of D₂O and said anysolvent.
 16. A composition comprising: at least one glycosaminoglycancomprising at least one saccharide comprising anomeric or target carbonexhibiting respective anomeric or target ¹³C chemical shift signal inthe range of about 55 ppm to about 115 ppm when analyzed by NMR; atleast one reference compound comprising at least one reference carbonatom that exhibits a reference ¹³C NMR chemical shift signal separatedfrom said anomeric or target ¹³C chemical shift signal, and saidreference ¹³C chemical shift signal exhibits a concentration dependentsignal intensity.
 17. The composition of claim 16, wherein saidreference ¹³C NMR chemical shift signal is in the range of about 25-27ppm.
 18. The composition of claim 16, wherein said reference ¹³C-NMRchemical shift signal is outside the range of 55 to 115 ppm and outsidethe range of 22-24 ppm.
 19. The composition of claim 16, wherein saidreference ¹³C-NMR chemical shift signal is a singlet within the range ofabout 25 ppm to about 27 ppm inclusive of the range limits.
 20. Thecomposition of claim 16, wherein said reference ¹³C-NMR chemical shiftis relative to the singlet chemical shift of3-(trimethylsilyl)-priopionic-D4 acid assigned as 0 ppm.
 21. Thecomposition of claim 16 further comprising a chemical shift referencestandard defining 0 ppm.
 22. The composition of claim 21 furthercomprising at least one deuterated solvent for said at least oneglycosaminoglycan, said at least one reference compound, and saidchemical shift reference standard.
 23. The composition of claim 22,wherein said deuterated solvent is selected from the group consisting ofD₂O, any solvent that will solubilize the at least one GAG and the atleast one reference compound, and a combination of D₂O and said anysolvent.
 24. A composition comprising at least one glycosaminoglycancomprising at least one saccharide comprising a) anomeric or targethydrogen exhibiting respective anomeric or target ¹H chemical shiftsignal in the range of about 3.2 ppm to about 6 ppm when analyzed byNMR; or b) anomeric or target carbon exhibiting respective anomeric ortarget ¹³C chemical shift signal in the range of about 55 ppm to about115 ppm when analyzed by NMR; wherein said glycosaminoglycan is anoligosaccharide or polysaccharide; and dimethylmalonic acid.
 25. Thecomposition of claim 24 further comprising a chemical shift referencestandard defining 0 ppm.
 26. The composition of claim 25 furthercomprising at least one deuterated solvent for said at least oneglycosaminoglycan, said at least one reference compound, and saidchemical shift reference standard.
 27. The composition of claim 24,wherein said at least one saccharide is selected from the groupconsisting of 4,5-unsaturated 2-O sulfo-uronic acid (ΔU2S),4,5-unsaturated uronic acid (ΔU), 2-N-sulfo-1,6-anhydroglucosamine(1,6-an.A), 2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M),2-N-sulfo-6-O-sulfoglucosamine (ANS6S), 2,5-anhydro mannitol,N-sulfoglucosamine, glucuronic acid, N-sulfo-6-O-sulfoglucosamine,2-O-sulfoiduronic acid, iduronic acid, N-sulfo-3-O-sulfoglucosamine,N-sulfo-3,6-di-O-sulfoglucosamine, galacturonic acid, Xylose,N-acetylglucosamine and N-acetyl-6-O-sulfoglucosamine.
 28. Thecomposition of claim 24, wherein said at least one glycosaminoglycan isselected from the group consisting of heparin, heparan, enoxaparin,bemiparin, dalteparin, tinzaparin, a salt of any of the preceding, aderivative of any of the preceding, a sulfated or non-sulfated form ofany of the preceding, an ultra-low molecular weight form of any of thepreceding, a low molecular weight form of any of the preceding, a highmolecular weight form of any of the preceding, an unfractionated form ofany of the preceding, a fractionated form of any of the preceding, and acombination of any thereof; and said at least one saccharide is selectedfrom the group consisting of 4,5-unsaturated 2-O sulfo-uronic acid(ΔU2S), 4,5-unsaturated uronic acid (ΔU),2-N-sulfo-1,6-anhydroglucosamine (1,6-an.A),2-N-sulfo-1,6-anhydro-mannosamine (1,6-an.M),2-N-sulfo-6-O-sulfoglucosamine (ANS6S), 2,5-anhydro mannitol,N-sulfoglucosamine, glucuronic acid, N-sulfo-6-O-sulfoglucosamine,2-O-sulfoiduronic acid, iduronic acid, N-sulfo-3-O-sulfoglucosamine,N-sulfo-3,6-di-O-sulfoglucosamine, galacturonic acid, Xylose,N-acetylglucosamine and N-acetyl-6-O-sulfoglucosamine; wherein saidglycosaminoglycan is an oligosaccharide or polysaccharide; and saidcomposition further comprises: dimethylmalonic acid;3-(trimethylsilyl)-priopionic-D4 acid; and at least one deuteratedsolvent for said at least one glycosaminoglycan, dimethylmalonic acid,and 3-(trimethylsilyl)-priopionic-D4 acid.